Sequences directed against hepatocyte growth factor (hfg) and polypeptides comprising the same for the treatment of cancers and/or tumors

ABSTRACT

The present invention relates to biological materials against HGF and more in particular to polypeptides, nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions and in particular to pharmaceutical compositions that comprise such polypeptides, for prophylactic, therapeutic or diagnostic purposes. In particular, the biological materials of the present invention inhibit binding of HGF to its receptor c-Met.

FIELD OF THE INVENTION

The present invention relates to biological materials related to HGF andmore in particular to polypeptides, nucleic acids encoding suchpolypeptides; to methods for preparing such polypeptides; to host cellsexpressing or capable of expressing such polypeptides; to compositionsand in particular to pharmaceutical compositions that comprise suchpolypeptides, for prophylactic, therapeutic or diagnostic purposes.

BACKGROUND

Receptor tyrosine kinases (RTKs) are key regulators of critical cellularprocesses such as cell growth, differentiation, neo-vascularization, andtissue repair. In addition to their importance in normal physiology,aberrant expression of certain RTKs has been implicated in thedevelopment and progression of many types of cancer. These RTKs haveemerged as promising drug targets for cancer therapy.

The RTK c-Met is the cell surface receptor for Hepatocyte Growth Factor(HGF), also known as scatter factor (Cooper et al. Nature 1984;311:29-33; Bottaro et al. Science 1991; 251:802-4). HGF is a 90 kDmultidomain glycoprotein that is highly related to members of theplasminogen serine protease family. Hepatocycte Growth Factor issecreted as a single-chain, inactive polypeptide by mesenchymal cells,and is cleaved by serine proteases into a 69-kDa alpha-chain and 34-kDabeta-chain. (Birchmeier et al., Nat Rev Mol Cell Biol 2003; 4:915-25).The α chain NH₂-terminal portion contains the high-affinity c-Metreceptor-binding domain, but the β chain is required to interact withthe c-Met receptor for receptor activation (Matsumoto & Nakamura Cancer3d 2003; 94:321-7). HGF is the only known ligand for the c-Met receptor(Birchmeier et al. Nat Rev Mol Cell Biol 2003; 4:915-25). The c-Metreceptor, like its ligand, is a disulfide-linked heterodimer consistingof extracellular α and β chains. The α chain, heterodimerized to theamino-terminal portion of the β chain, forms the major ligand-bindingsite in the extracellular domain. The carboxy-terminal tail of c-Metincludes tyrosines Y1349 and Y1356, which, when phosphorylated, serve asdocking sites for intracellular adaptor proteins, leading to downstreamsignaling (Ponzetto et al. Mol Cell Biol 1993; 13:4600-8). The c-Met/HGFpathway is the main driver of the invasive growth program, a series ofevents including cell proliferation, scattering, migration, survival,and invasion of tissues. Under normal circumstances, the invasive growthprogram is essential for correct organ formation during embryogenesisand in adult homeostasis. Importantly, it is also involved intumorigenesis, tumor angiogenesis and metastasis. The c-Met receptor isexpressed in the epithelial cells of many organs during embryogenesisand also in adulthood, like liver, prostate, pancreas, muscle, kidneyand bone marrow. In tumor cells, c-Met activation triggers diverseseries of signaling cascades resulting in cell growth, proliferation,invasion, metastasis formation and escape from apoptosis. Overexpressionof HGF and c-Met is indicative of increased aggressiveness of tumors andpoor prognostic outcome of cancer patients. HGF and c-Met expressionhave been observed in most solid tumors, including; head and neck,bladder, breast, cervical, colorectal, gastric, liver, lung, ovarian,pancreatic, prostate, renal and thyroid cancers.

SUMMARY OF THE INVENTION

Targeting the HGF/c-Met pathway provides a therapeutic opportunity.Preventing ligand/receptor binding would result in growth inhibition andtumor regression by inhibiting proliferation and enhancing apoptosis.Since HGF, which is also known as scatter factor, is more elusivecompared to the membrane bound receptor c-Met, most studies have focusedon the receptor. Indeed, one-armed 5D5 (OA5D5, MetMAb; Genentech) is ahumanized, monovalent, antagonistic anti-c-Met antibody derived from theagonistic monoclonal antibody 5D5 (Nguyen et al. Cancer Gene Ther 2003;10:840-9).

On the other hand, Cao et al needed a combination of 3 monoclonalantibodies to achieve neutralizing activity to HGF in glioma xenografttumors, and suggested that the complex heterodimeric structure of HGFmakes it necessary to simultaneously target multiple HGF epitopes bycombining mAbs (Cao et al., Proc Natl Acad Sci USA 2001; 98:7443-8).

AMG102 (rilotumumab; Amgen, Inc.) was identified in an extensive screen,resulting in 3 potential candidates, of which each recognized adifferent epitope. Although AMG102 had intermediate affinity for HGF (asjudged by binding affinity), it was the only mAb identified thatcompletely blocked the binding of HGF to c-Met (Kim et al. 2006 ClinCancer Res 12:1292-1298).

Several lines of evidence indicate that the HGF/c-Met pathway is also atherapeutic target in metastatic renal cell carcinoma (mRCC).Nevertheless, Schöffski et al. demonstrate that no significant growthinhibition occurred with AMG102 (Schöffski et al. 2010 BJU Intdoi:10.1111/j.1464). Similarly, HGF and its receptor c-Met have beenimplicated in the pathogenesis of glioblastoma (GBM), but Wen andcolleagues showed in a phase II study that AMG102 monotherapy treatmentat doses up to 20 mg/kg was not associated with significant antitumoractivity in the selected patient groups (Wen et al. 2011 Neuro-Oncologydoi:10.1093/neuonc/noq198).

Indeed, according to the current biomedical understanding, drugresistance is caused by a complex network of proteins responsible forthe regulation of cell proliferation, apoptosis, migration and invasion.Currently, no systematic description of growth factor receptor dependentsignaling pathways is available. The molecular pathways by whichHGF/c-Met abnormalities drive cancer development are extremely complexand involve many interconnected signaling pathways, including bothsignaling molecules (such as Ras and PI3K), receptors (such as EGFR),and growth factors (such as VEGF).

Targeting serum albumin to extend the half-life of biological moleculessuch as e.g., immunoglobulin single variable domains has been describede.g., in WO2008/028977, WO04/041865 and WO08/122,787.

The art is in need of effective and/or more potent HGF antagonistshaving superior selectivity and specificity over small molecule drugs,an ability to modulate half-life, and/or a superior tumour targeting,e.g., are smaller than conventional antibodies and have an albumin-basedtumour targeting strategy. Furthermore, the art is in need ofdiagnostically, preventatively, and/or therapeutically suitable HGFantagonists such as provided herein.

The present invention relates to an immunoglobulin single variabledomain that can bind (to) HGF (SEQ ID NO: 1) with a Kd of less than 50nM. In an embodiment, said immunoglobulin single variable domain caninhibit binding of HGF, preferably human HGF (SEQ ID NO: 1) to c-Met,preferably human c-Met (SEQ ID NO: 4), with a Kd of less than 50 nM, andoptionally a maximal HGF displacement level of 60% to 80% or more. Inparticular, wherein the immunoglobulin single variable domain comprisesan amino acid sequence of formula 1: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1);wherein FR1 to FR4 refer to framework regions 1 to 4 and are frameworkregions (FR5) of an immunoglobulin single variable domain; and whereinCDR1 is chosen from the group consisting of: SEQ ID NOs: 40-51,polypeptides that have at least 80% amino acid identity with SEQ ID NOs:40-51, and polypeptides that have 3, 2, or 1 amino acid difference withSEQ ID NOs: 40-51; and wherein CDR2 is chosen from the group consistingof: SEQ ID NOs: 64-75; polypeptides that have at least 80% amino acididentity with SEQ ID NOs: 64-75; and polypeptides that have 3, 2, or 1amino acid difference with SEQ ID NOs: 64-75; and wherein CDR3 is chosenfrom the group consisting of: SEQ ID NOs: 88-99; polypeptides that haveat least 80% amino acid identity with at least one of the immunoglobulinsingle variable domains of SEQ ID NOs: 88-99; and polypeptides that have3, 2, or 1 amino acid difference with SEQ ID NOs: 88-99; even morepreferably, wherein the framework regions (FR5) have a sequence identityof more than 80% with the FRs of SEQ ID NOs: 7-25.

The present invention further relates to an immunoglobulin singlevariable domain as described herein, wherein the immunoglobulin singlevariable domain comprises an amino acid sequence with the formula 1:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1); wherein FR1 to FR4 refer toframework regions 1 to 4 and are framework regions of an immunoglobulinsingle variable domain; wherein CDR1 is SEQ ID NO: 40, wherein CDR2 isSEQ ID NO: 64, 67, 69 or 72; and wherein CDR3 is SEQ ID NO: 88, 91, 93or 96; wherein CDR1 is SEQ ID NO: 40, wherein CDR2 is SEQ ID NO: 64; andwherein CDR3 is SEQ ID NO: 88; or wherein CDR1 is SEQ ID NO: 45, whereinCDR2 is SEQ ID NO: 69; and wherein CDR3 is SEQ ID NO: 93.

The present invention also relates to a polypeptide comprising animmunoglobulin single variable domain as described herein; preferablywherein the polypeptide is selected from the group consisting ofpolypeptides that have an amino acid sequence with a sequence identityof more than 80% with SEQ ID NOs: 7 to 25; even more preferably whereinthe polypeptide is selected from the group consisting of polypeptidesthat have an amino acid sequence with a sequence identity of more than80% with SEQ ID NOs: 7 or 18; even more preferably additionallycomprising an immunoglobulin single variable domain binding human serumalbumin such as e.g. Alb8 (SEQ ID NO: 115) or Alb11 (SEQ ID NO: 114).

In addition, the present invention relates to an immunoglobulin singlevariable domain as described herein or the polypeptide as describedherein, wherein the IC50 in an Alphascreen® assay is 30 nM or lower, oreven wherein the IC50 in an Alphascreen® assay is 3 nM or lower. Thepresent invention also relates to a nucleic acid sequence encoding i)for an immunoglobulin single variable domain as described herein; or ii)for a polypeptide as described herein.

Moreover, the present invention relates to a pharmaceutical compositioncomprising i) for an immunoglobulin single variable domain as describedherein; or ii) for a polypeptide as described herein; and optionally apharmaceutically acceptable excipient.

Additionally, the present invention relates to an immunoglobulin singlevariable domain as described herein; or ii) for a polypeptide asdescribed herein, for use in treating cancer.

Also, the present invention relates to a method for producing animmunoglobulin single variable domain as described herein; or ii) for apolypeptide as described herein, said method at least comprising thesteps of: (a) expressing, in a suitable host cell or host organism or inanother suitable expression system, a nucleic acid or nucleotidesequence as described herein; optionally followed by (b) isolatingand/or purifying said immunoglobulin single variable domain or saidpolypeptide.

The present invention also relates to a method for screeningimmunoglobulin single variable domains directed against HGF and inparticular human HGF (SEQ ID NO: 1) that comprises at least the steps of(a) providing a set, collection or library of immunoglobulin singlevariable domains; and (b) screening said set, collection or library ofimmunoglobulin single variable domains for immunoglobulin singlevariable domains that can bind to and/or have affinity for HGF and inparticular human HGF (SEQ ID NO: 1); and (c) isolating the amino acidsequence(s) that can bind to and/or have affinity for HGF and inparticular human HGF (SEQ ID NO: 1).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 serum titrations of llama 85 and 092

FIG. 2 Screen of periplasmic extracts using AlphaScreen™. The individualclones show a reduced signal when the HGF/c-Met interaction is blocked.

FIG. 3 aligned sequences of the anti-HGF lead Nanobodies. The cysteineresidues are boxed.

FIG. 4 IC50 determination using AlphaScreen™.

FIG. 5 Biodistribution of αHGF Nanobodies ⁸⁹Zr-1E2-ALB (A) and⁸⁹Zr-6E10-ALB (B) in nude mice bearing U87 MG xenografts at 24, 48, 72and 168 h p.i.

FIG. 6 Biodistribution of αHGF Nanobody 1E2-ALB labeled with theresidualizing PET isotope ⁸⁹Zr or the non residualizing radio isotope¹³¹I, in nude mice bearing U87 MG xenografts at 72 h p.i.

FIG. 7 Bloodkinetics of ⁸⁹Zr labeled αHGF Nanobodies.

FIG. 8 (A) Therapy study with αHGF Nanobodies in nude mice bearing U87MG glioblastoma xenografts. Treatment was 3 times a week for 5 weeks.Kaplan Meier survival curves of nude mice treated with different amountsof 1E2-ALB (B) or 6E10-ALB (C). Treatment with all Nanobodyconcentrations caused significant regression of the established tumorsafter day 6 (p<0.01), and curative responses after treatment with 30 or100 μg 1E2-ALB, or 100 μg 6E10-ALB.

DESCRIPTION OF THE INVENTION

Immunoglobulin sequences, such as antibodies and antigen bindingfragments derived there from (e.g., immunoglobulin single variabledomains) are used to specifically target their respective antigens inresearch and therapeutic applications. The generation of immunoglobulinsingle variable domains such as e.g., VHHs may involve the immunizationof an experimental animal such as a Llama, construction of phagelibraries from immune tissue, selection of phage displaying antigenbinding immunoglobulin single variable domains and screening of saiddomains and engineered constructs thereof for the desired specificities(WO 94/04678). Alternatively, similar immunoglobulin single variabledomains such as e.g., dAbs can be generated by selecting phagedisplaying antigen binding immunoglobulin single variable domainsdirectly from naive or synthetic libraries and subsequent screening ofsaid domains and engineered constructs thereof for the desiredspecificities (Ward et al., Nature, 1989, 341: 544-6; Holt et al.,Trends Biotechnol., 2003, 21(11):484-490; as well as for example WO06/030220, WO 06/003388 and other published patent applications ofDomantis Ltd.). Unfortunately, the use of monoclonal and/or heavilyengineered antibodies also carries a high manufacturing cost and mayresult in suboptimal tumor penetration compared to other strategies.

The present invention relates to particular polypeptides, also referredto as “polypeptides of the invention” or “immunoglobulin single variabledomain of the invention” or “ISVD of the invention” that comprise or,more preferably, essentially consist of (i) a first building blockconsisting essentially of one or more (preferably one) immunoglobulinsingle variable domain(s), wherein said immunoglobulin single variabledomain(s) is (are) directed against HGF and in particular against humanHGF; (ii) optionally a second building block consisting essentially ofone or more (preferably one) immunoglobulin single variable domain(s),wherein said immunoglobulin single variable domain(s) is (are) directedagainst serum albumin and in particular against human serum albumin (andeven more preferably wherein said immunoglobulin single variable domainis Alb8 or Alb11 (as herein defined)); (iii) optionally a third and/orfourth building block consisting essentially of one or moreimmunoglobulin single variable domain(s), wherein said immunoglobulinsingle variable domain(s) is (are) directed against EGFR, in particularhuman EGFR (hEGFR), and/or is (are) directed against VEGF, in particularhuman VEGF (hVEGF). Furthermore, the invention also relates to nucleicacids encoding such polypeptides; to methods for preparing suchpolypeptides; to host cells expressing or capable of expressing suchpolypeptides; to compositions and in particular to pharmaceuticalcompositions that comprise such polypeptides, nucleic acids and/or hostcells; and to uses of such polypeptides, nucleic acids, host cellsand/or compositions for prophylactic, therapeutic or diagnosticpurposes. Other aspects, embodiments, advantages and applications of theinvention will become clear from the further description herein.

In this study, two αHGF Nanobodies 1E2-ALB and 6E10-ALB were developedand characterized for their potential in diagnosis and therapy ofcancer. After labeling with the positron emitter Zirconium-89 theNanobodies were evaluated in biodistribution studies in nude micebearing U87 MG glioblastoma xenografts. Besides that, αHGF-Nanobodieswere tested as therapeutic agents by inhibiting the binding of HGF tothe c-Met receptor in the same mouse model.

DEFINITIONS

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks mentioned in    paragraph a) on page 46 of WO 08/020,079.-   b) Unless indicated otherwise, the term “immunoglobulin single    variable domain” or “ISVD” is used as a general term to include but    not limited to antigen-binding domains or fragments such as V_(HH)    domains or V_(H) or V_(L) domains, respectively. The terms    antigen-binding molecules or antigen-binding protein are used    interchangeably and include also the term Nanobodies. The    immunoglobulin single variable domains can be light chain variable    domain sequences (e.g., a V_(L)-sequence), or heavy chain variable    domain sequences (e.g., a V_(H)-sequence); more specifically, they    can be heavy chain variable domain sequences that are derived from a    conventional four-chain antibody or heavy chain variable domain    sequences that are derived from a heavy chain antibody. Accordingly,    the immunoglobulin single variable domains can be domain antibodies,    or immunoglobulin sequences that are suitable for use as domain    antibodies, single domain antibodies, or immunoglobulin sequences    that are suitable for use as single domain antibodies, “dAbs”, or    immunoglobulin sequences that are suitable for use as dAbs, or    Nanobodies, including but not limited to V_(HH) sequences. The    invention includes immunoglobulin sequences of different origin,    comprising mouse, rat, rabbit, donkey, human and camelid    immunoglobulin sequences. The immunoglobulin single variable domain    includes fully human, humanized, otherwise sequence optimized or    chimeric immunoglobulin sequences. The immunoglobulin single    variable domain and structure of an immunoglobulin single variable    domain can be considered—without however being limited thereto—to be    comprised of four framework regions or “FR's”, which are referred to    in the art and herein as “Framework region 1” or “FR1”; as    “Framework region 2” or “FR2”; as “Framework region 3” or “FR3”; and    as “Framework region 4” or “FR4”, respectively; which framework    regions are interrupted by three complementary determining regions    or “CDR's”, which are referred to in the art as “Complementarity    Determining Region 1” or “CDR1”; as “Complementarity Determining    Region 2” or “CDR2”; and as “Complementarity Determining Region 3”    or “CDR3”, respectively. It is noted that the terms Nanobody or    Nanobodies are registered trademarks of Ablynx N.V. and thus may    also be referred to as Nanobody® or Nanobodies®, respectively.-   c) Unless indicated otherwise, the terms “immunoglobulin sequence”,    “sequence”, “nucleotide sequence” and “nucleic acid” are as    described in paragraph b) on page 46 of WO 08/020,079.-   d) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein; as well as to    for example the following reviews Presta, Adv. Drug Deliv. Rev.    2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):    49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45;    Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et    al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for    protein engineering, such as affinity maturation and other    techniques for improving the specificity and other desired    properties of proteins such as immunoglobulins.-   e) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code. Reference is made to    Table A-2 on page 48 of the International application WO 08/020,079    of Ablynx N.V. entitled “Immunoglobulin single variable domains    directed against IL-6R and polypeptides comprising the same for the    treatment of diseases and disorders associated with Il-6 mediated    signalling”.-   f) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated or    determined as described in paragraph e) on page 49 of WO 08/020,079    (incorporated herein by reference), such as by dividing [the number    of nucleotides in the first nucleotide sequence that are identical    to the nucleotides at the corresponding positions in the second    nucleotide sequence] by [the total number of nucleotides in the    first nucleotide sequence] and multiplying by [100%], in which each    deletion, insertion, substitution or addition of a nucleotide in the    second nucleotide sequence—compared to the first nucleotide    sequence—is considered as a difference at a single nucleotide    (position); or using a suitable computer algorithm or technique,    again as described in paragraph e) on pages 49 of WO 08/020,079    (incorporated herein by reference).-   g) For the purposes of comparing two or more immunoglobulin single    variable domains or other amino acid sequences such e.g. the    polypeptides of the invention etc., the percentage of “sequence    identity” between a first amino acid sequence and a second amino    acid sequence (also referred to herein as “amino acid identity”) may    be calculated or determined as described in paragraph f) on pages 49    and 50 of WO 08/020,079 (incorporated herein by reference), such as    by dividing [the number of amino acid residues in the first amino    acid sequence that are identical to the amino acid residues at the    corresponding positions in the second amino acid sequence] by [the    total number of amino acid residues in the first amino acid    sequence] and multiplying by [100%], in which each deletion,    insertion, substitution or addition of an amino acid residue in the    second amino acid sequence—compared to the first amino acid    sequence—is considered as a difference at a single amino acid    residue (position), i.e., as an “amino acid difference” as defined    herein; or using a suitable computer algorithm or technique, again    as described in paragraph f) on pages 49 and 50 of WO 08/020,079    (incorporated herein by reference).

Also, in determining the degree of sequence identity between twoimmunoglobulin single variable domains, the skilled person may take intoaccount so-called “conservative” amino acid substitutions, as describedon page 50 of WO 08/020,079.

Any amino acid substitutions applied to the polypeptides describedherein may also be based on the analysis of the frequencies of aminoacid variations between homologous proteins of different speciesdeveloped by Schulz et al., Principles of Protein Structure,Springer-Verlag, 1978, on the analyses of structure forming potentialsdeveloped by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv.Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicitypatterns in proteins developed by Eisenberg et al., Proc. Natl. Acad.Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J. Molec. Biol. 157:105-132, 198 1, and Goldman et al., Ann. Rev. Biophys. Chem. 15:321-353, 1986, all incorporated herein in their entirety by reference.Information on the primary, secondary and tertiary structure ofNanobodies is given in the description herein and in the generalbackground art cited above. Also, for this purpose, the crystalstructure of a V_(HH) domain from a llama is for example given byDesmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996);Spinelli et al., Natural Structural Biology (1996); 3, 752-757; andDecanniere et al., Structure, Vol. 7, 4, 361 (1999). Further informationabout some of the amino acid residues that in conventional V_(H) domainsform the V_(H)/V_(L) interface and potential camelizing substitutions onthese positions can be found in the prior art cited above.

-   h) Immunoglobulin single variable domains and nucleic acid sequences    are said to be “exactly the same” if they have 100% sequence    identity (as defined herein) over their entire length.

When comparing two immunoglobulin single variable domains, the term“amino acid difference” refers to an insertion, deletion or substitutionof a single amino acid residue on a position of the first sequence,compared to the second sequence; it being understood that twoimmunoglobulin single variable domains can contain one, two or more suchamino acid differences.

-   j) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this has the meaning given in    paragraph i) on pages 51-52 of WO 08/020,079.-   k) The term “in essentially isolated form” has the meaning given to    it in paragraph j) on pages 52 and 53 of WO 08/020,079.-   l) The terms “domain” and “binding domain” have the meanings given    to it in paragraph k) on page 53 of WO 08/020,079.-   m) The terms “antigenic determinant” and “epitope”, which may also    be used interchangeably herein, have the meanings given to it in    paragraph l) on page 53 of WO 08/020,079.-   n) As further described in paragraph m) on page 53 of WO 08/020,079,    an amino acid sequence (such as an antibody, a polypeptide of the    invention, or generally an antigen binding protein or polypeptide or    a fragment thereof) that can (specifically) bind to, that has    affinity for and/or that has specificity for a specific antigenic    determinant, epitope, antigen or protein (or for at least one part,    fragment or epitope thereof) is said to be “against” or “directed    against” said antigenic determinant, epitope, antigen or protein.-   o) The term “specificity” has the meaning given to it in    paragraph n) on pages 53-56 of WO 08/020,079; and as mentioned    therein refers to the number of different types of antigens or    antigenic determinants to which a particular antigen-binding    molecule or antigen-binding protein (such as a polypeptide of the    invention) molecule can bind. The specificity of an antigen-binding    protein can be determined based on affinity and/or avidity, as    described on pages 53-56 of WO 08/020,079 (incorporated herein by    reference), which also describes some preferred techniques for    measuring binding between an antigen-binding molecule (such as a    polypeptide or ISVD of the invention) and the pertinent antigen.    Typically, antigen-binding proteins (such as the immunoglobulin    single variable domains, and/or polypeptides of the invention) will    bind to their antigen with a dissociation constant (K_(D)) of 10⁻⁵    to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²    moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter    (i.e., with an association constant (K_(A)) of 10⁵ to 10¹²    liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or more    and more preferably 10⁸ to 10¹² liter/moles). Any K_(D) value    greater than 10⁻⁴ mol/liter (or any K_(A) value lower than 10⁴    liter/mol) is generally considered to indicate non-specific binding.    Preferably, a monovalent immunoglobulin single variable domain of    the invention will bind to the desired antigen with an affinity less    than 500 nM, preferably less than 200 nM, more preferably less than    10 nM, such as less than 500 pM. Specific binding of an    antigen-binding protein to an antigen or antigenic determinant can    be determined in any suitable manner known per se, including, for    example, Scatchard analysis and/or competitive binding assays, such    as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich    competition assays, and the different variants thereof known per se    in the art; as well as the other techniques mentioned herein. As    will be clear to the skilled person, and as described on pages 53-56    of WO 08/020,079, the dissociation constant may be the actual or    apparent dissociation constant. Methods for determining the    dissociation constant will be clear to the skilled person, and for    example include the techniques mentioned on pages 53-56 of WO    08/020,079.-   p) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as described in    paragraph o) on page 57 of WO 08/020,079 and as mentioned therein    refers to the time taken for the serum concentration of the amino    acid sequence, compound or polypeptide to be reduced by 50%, in    vivo, for example due to degradation of the sequence or compound    and/or clearance or sequestration of the sequence or compound by    natural mechanisms. The in vivo half-life of an amino acid sequence,    compound or polypeptide of the invention can be determined in any    manner known per se, such as by pharmacokinetic analysis. Suitable    techniques will be clear to the person skilled in the art, and may    for example generally be as described in paragraph o) on page 57 of    WO 08/020,079. As also mentioned in paragraph o) on page 57 of WO    08/020,079, the half-life can be expressed using parameters such as    the t½-alpha, t½-beta and the area under the curve (AUC). Reference    is for example made to the Experimental Part below, as well as to    the standard handbooks, such as Kenneth, A et al: Chemical Stability    of Pharmaceuticals: A Handbook for Pharmacists and Peters et al,    Pharmacokinete analysis: A Practical Approach (1996). Reference is    also made to “Pharmacokinetics”, M Gibaldi & D Perron, published by    Marcel Dekker, 2nd Rev. edition (1982). The terms “increase in    half-life” or “increased half-life” as also as defined in    paragraph o) on page 57 of WO 08/020,079 and in particular refer to    an increase in the t½-beta, either with or without an increase in    the t½-alpha and/or the AUC or both.-   q) In respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerisation (such as    homomerization or heterodimerization) of the target or antigen; or    any other site, epitope, antigenic determinant, part, domain or    stretch of amino acid residues on the target or antigen that is    involved in a biological action or mechanism of the target or    antigen. More generally, an “interaction site” can be any site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen to which an amino acid    sequence or polypeptide of the invention can bind such that the    target or antigen (and/or any pathway, interaction, signalling,    biological mechanism or biological effect in which the target or    antigen is involved) is modulated (as defined herein).-   r) An immunoglobulin single variable domain or polypeptide is said    to be “specific for” a first target or antigen compared to a second    target or antigen when it binds to the first antigen with an    affinity/avidity (as described above, and suitably expressed as a    K_(D) value, K_(A) value, K_(off) rate and/or K_(on) rate) that is    at least 10 times, such as at least 100 times, and preferably at    least 1000 times, and up to 10.000 times or more better than the    affinity with which said amino acid sequence or polypeptide binds to    the second target or polypeptide. For example, the first antigen may    bind to the target or antigen with a K_(D) value that is at least 10    times less, such as at least 100 times less, and preferably at least    1000 times less, such as 10.000 times less or even less than that,    than the K_(D) with which said amino acid sequence or polypeptide    binds to the second target or polypeptide. Preferably, when an    immunoglobulin single variable domain or polypeptide is “specific    for” a first target or antigen compared to a second target or    antigen, it is directed against (as defined herein) said first    target or antigen, but not directed against said second target or    antigen.-   s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are    used interchangeably herein to mean the ability of an immunoglobulin    single variable domain or polypeptide to interfere with the binding    of the natural ligand HGF to c-Met or with the binding of the    natural ligand EGF to EGFR, or with the binding of the natural    ligand VEGF to VEGF receptors (such as VEGFR-1R (Flt-1), VEGFR-2    (KDR/Flk-1) and/or VEGFR-3 (Flt-4)), respectively. The extent to    which an immunoglobulin single variable domain or polypeptide of the    invention is able to interfere with the binding of another compound    such as the natural ligand to its target, e.g., c-Met, VEGF or EGFR,    and therefore whether it can be said to cross-block according to the    invention, can be determined using competition binding assays. One    particularly suitable quantitative cross-blocking assay uses a FACS-    or an ELISA-based approach or Alphascreen to measure competition    between the labelled (e.g., His tagged or biotinylated)    immunoglobulin single variable domain or polypeptide according to    the invention and the other binding agent in terms of their binding    to the target. The experimental part generally describes suitable    FACS-, ELISA- or Alphascreen-displacement-based assays for    determining whether a binding molecule cross-blocks or is capable of    cross-blocking an immunoglobulin single variable domain or    polypeptide according to the invention. It will be appreciated that    the assay can be used with any of the immunoglobulin single variable    domains or other binding agents described herein. Thus, in general,    a cross-blocking amino acid sequence or other binding agent    according to the invention is for example one which will bind to the    target in the above cross-blocking assay such that, during the assay    and in the presence of a second amino acid sequence or other binding    agent of the invention, the recorded displacement of the    immunoglobulin single variable domain or polypeptide according to    the invention is between 60% and 100% (e.g., in ELISA/Alphascreen    based competition assay) or between 80% to 100% (e.g., in FACS based    competition assay) of the maximum theoretical displacement (e.g.    displacement by cold (e.g., unlabeled) immunoglobulin single    variable domain or polypeptide that needs to be cross-blocked) by    the to be tested potentially cross-blocking agent that is present in    an amount of 0.01 mM or less (cross-blocking agent may be another    conventional monoclonal antibody such as IgG, classic monovalent    antibody fragments (Fab, scFv)) and engineered variants (e.g.,    diabodies, triabodies, minibodies, VHHs, dAbs, VHs, VLs).-   t) An amino acid sequence such as e.g. an immunoglobulin single    variable domain or polypeptide according to the invention is said to    be a “VHH1 type immunoglobulin single variable domain” or “VHH type    1 sequence”, if said VHH1 type immunoglobulin single variable domain    or VHH type 1 sequence has 85% identity (using the VHH1 consensus    sequence as the query sequence and use the blast algorithm with    standard setting, i.e., blosom62 scoring matrix) to the VHH1    consensus sequence (SEQ ID NO: 127:    QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAP-GKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA),    and mandatorily has a cysteine in position 50, i.e., C50 (using    Kabat numbering).-   u) An amino acid sequence such as e.g., an immunoglobulin single    variable domain or polypeptide according to the invention is said to    be “cross-reactive” for two different antigens or antigenic    determinants (such as serum albumin from two different species of    mammal, such as human serum albumin and cynomolgus monkey serum    albumin) if it is specific for (as defined herein) both these    different antigens or antigenic determinants.-   v) As further described in paragraph q) on pages 58 and 59 of WO    08/020,079 (incorporated herein by reference), the amino acid    residues of an immunoglobulin single variable domain are numbered    according to the general numbering for V_(H) domains given by Kabat    et al. (“Sequence of proteins of immunological interest”, US Public    Health Services, NIH Bethesda, Md., Publication No. 91), as applied    to V_(HH) domains from Camelids in the article of Riechmann and    Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195    (see for example FIG. 2 of this publication), and accordingly FR1 of    an immunoglobulin single variable domain comprises the amino acid    residues at positions 1-30, CDR1 of an immunoglobulin single    variable domain comprises the amino acid residues at positions    31-35, FR2 of an immunoglobulin single variable domain comprises the    amino acids at positions 36-49, CDR2 of an immunoglobulin single    variable domain comprises the amino acid residues at positions    50-65, FR3 of an immunoglobulin single variable domain comprises the    amino acid residues at positions 66-94, CDR3 of an immunoglobulin    single variable domain comprises the amino acid residues at    positions 95-102, and FR4 of an immunoglobulin single variable    domain comprises the amino acid residues at positions 103-113.-   w) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

Polypeptides of the Invention and Uses Thereof

The polypeptides of the present invention can generally be used tomodulate, and in particular inhibit and/or prevent, binding of HGF andin particular human HGF (SEQ ID NO: 1; Swiss Prot database: P14210) toc-Met and in particular human c-Met (SEQ ID NO: 4), and thus tomodulate, and in particular inhibit or prevent, the signalling that ismediated by c-Met and in particular human c-Met (SEQ ID NO: 4) and/orHGF and in particular human HGF (Swiss Prot database: P14210), tomodulate the biological pathways in which HGF and in particular humanHGF (SEQ ID NO: 1) and/or c-Met and in particular human c-Met areinvolved, and/or to modulate the biological mechanisms, responses andeffects associated with such signalling or these pathways (α-HGFbuilding blocks).

As such, the polypeptides and compositions of the present invention canbe used for the diagnosis, prevention and treatment of diseases anddisorders of the present invention (herein also “diseases and disordersof the present invention”) which include, but are not limited to cancer,e.g., carcinomas, gliomas, mesotheliomas, melanomas, lymphomas,leukemias, adenocarcinomas: breast cancer, ovarian cancer, cervicalcancer, glioblastoma, multiple myeloma (including monoclonal gammopathyof undetermined significance, asymptomatic and symptomatic myeloma),prostate cancer, and Burkitt's lymphoma, head and neck cancer, coloncancer, colorectal cancer, non-small cell lung cancer, small cell lungcancer, cancer of the esophagus, stomach cancer, pancreatic cancer,hepatobiliary cancer, cancer of the gallbladder, cancer of the smallintestine, rectal cancer, kidney cancer, bladder cancer, prostatecancer, penile cancer, urethral cancer, testicular cancer, vaginalcancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenalcancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skincancer, retinoblastomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma,Kaposi's sarcoma, multicentric Castleman's disease or AIDS-associatedprimary effusion lymphoma, neuroectodermal tumors, rhabdomyosarcoma (seee.g., Cancer, Principles and practice (DeVita, V. T. et al. eds 1997)for additional cancers); as well as any metastasis of any of the abovecancers, as well as non-cancer indications such as nasal polyposis; aswell as other disorders and diseases described herein. In particular,the polypeptides and compositions of the present invention can be usedfor the diagnosis, prevention and treatment of diseases involving HGFmediated metastasis, chemotaxis, cell adhesion, trans endothelialmigration, cell proliferation and/or survival, in particular non-smallcell lung cancer and multiple myeloma.

Generally, said “diseases and disorders of the present invention” can bedefined as diseases and disorders that can be diagnosed, preventedand/or treated, respectively, by suitably administering to a subject inneed thereof (i.e., having the disease or disorder or at least onesymptom thereof and/or at risk of attracting or developing the diseaseor disorder) of either a polypeptide or composition of the invention(and in particular, of a pharmaceutically active amount thereof) and/orof a known active principle active against HGF and in particular humanHGF (SEQ ID NO: 1) or a biological pathway or mechanism in which HGF andin particular human HGF (SEQ ID NO: 1) is involved (and in particular,of a pharmaceutically active amount thereof).

In particular, the polypeptides of the present invention can be used forthe diagnosis, prevention and treatment of diseases and disorders of thepresent invention which are characterized by excessive and/or unwantedHGF and in particular human HGF (SEQ ID NO: 1) signalling mediated byc-Met and in particular human c-Met or by the pathway(s) in which c-Metand in particular human c-Met is involved (e.g. HGF/c-Met axis).Examples of such diseases and disorders of the present invention willagain be clear to the skilled person based on the disclosure herein.

Thus, without being limited thereto, the immunoglobulin single variabledomains and polypeptides of the invention can for example be used todiagnose, prevent and/or to treat all diseases and disorders that arecurrently being diagnosed, prevented or treated with active principlesthat can modulate HGF and in particular human HGF (SEQ ID NO:1)-mediated signalling, such as those mentioned in the diseases andprior art cited above. It is also envisaged that the polypeptides of theinvention can be used to diagnose, prevent and/or to treat all diseasesand disorders for which treatment with such active principles iscurrently being developed, has been proposed, or will be proposed ordeveloped in the future. In addition, it is envisaged that, because oftheir favourable properties as further described herein, thepolypeptides of the present invention may be used for the diagnosis,prevention and treatment of other diseases and disorders than those forwhich these known active principles are being used or will be proposedor developed; and/or that the polypeptides of the present invention mayprovide new methods and regimens for treating the diseases and disordersdescribed herein.

Other applications and uses of the immunoglobulin single variabledomains and polypeptides of the invention will become clear to theskilled person from the further disclosure herein.

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as compositions comprising the same, that can beused in the diagnosis, prevention and/or treatment of diseases and/ordisorders of the invention; and to provide methods for the diagnosis,prevention and/or treatment of such diseases and disorders that involvethe administration and/or use of such agents and compositions.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions and/or methods that havecertain advantages compared to the agents, compositions and/or methodsthat are currently used and/or known in the art. These advantages willbecome clear from the further description below.

More in particular, it is an object of the invention to providetherapeutic proteins that can be used as pharmacologically activeagents, as well as compositions comprising the same, for the diagnosis,prevention and/or treatment of diseases and/or disorders of theinvention and of the further diseases and disorders mentioned herein;and to provide methods for the diagnosis, prevention and/or treatment ofsuch diseases and disorders that involve the administration and/or theuse of such therapeutic proteins and compositions.

Accordingly, it is a specific object of the present invention to provideimmunoglobulin single variable domains that are directed against HGF, inparticular against HGF from a warm-blooded animal, more in particularagainst HGF from a mammal such as e.g. mouse, and especially againsthuman HGF (SEQ ID NO: 1); and to provide proteins and polypeptidescomprising or essentially consisting of at least one such immunoglobulinsingle variable domain.

In particular, it is a specific object of the present invention toprovide such immunoglobulin single variable domains and such proteinsand/or polypeptides that are suitable for prophylactic, therapeuticand/or diagnostic use in a warm-blooded animal, and in particular in amammal, and more in particular in a human being.

More in particular, it is a specific object of the present invention toprovide such immunoglobulin single variable domains and such proteinsand/or polypeptides that can be used for the prevention, treatment,alleviation and/or diagnosis of one or more diseases, disorders orconditions associated with HGF and/or mediated by HGF (such as thediseases, disorders and conditions mentioned herein) in a warm-bloodedanimal, in particular in a mammal, and more in particular in a humanbeing.

It is also a specific object of the invention to provide suchimmunoglobulin single variable domains and such proteins and/orpolypeptides that can be used in the preparation of pharmaceutical orveterinary compositions for the prevention and/or treatment of one ormore diseases, disorders or conditions associated with and/or mediatedby HGF (such as the diseases, disorders and conditions mentioned herein)in a warm-blooded animal, in particular in a mammal, and more inparticular in a human being.

In the invention, generally, these objects are achieved by the use ofthe immunoglobulin single variable domains, proteins, polypeptides andcompositions that are described herein.

In general, the invention provides immunoglobulin single variabledomains that are directed against (as defined herein) and/or canspecifically bind (as defined herein) to HGF and in particular human HGF(SEQ ID NO: 1); as well as compounds and constructs, and in particularproteins and polypeptides, that comprise at least one such amino acidsequence or immunoglobulin single variable domain.

More in particular, the invention provides immunoglobulin singlevariable domains and polypeptides that can bind to HGF and in particularhuman HGF (SEQ ID NO: 1) with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a 1k_(off)-rate, as well as compoundsand constructs, and in particular proteins and polypeptides, thatcomprise at least one such amino acid sequence or immunoglobulin singlevariable domain.

Also, the immunoglobulin single variable domains and polypeptides thatcan bind to HGF and in particular human HGF (SEQ ID NO: 1) may becharacterized by biological potency, suitably measured and/or expressedas an IC₅₀ value, as further described and defined herein, for instance,such as by Alphascreen; as well as compounds and constructs, and inparticular proteins and polypeptides, that comprise at least one suchamino acid sequence or immunoglobulin single variable domain.

In particular aspect, the immunoglobulin single variable domains and/orpolypeptides of the invention:

-   -   bind to human HGF (SEQ ID NO: 1) with an IC50 of 50 nM or lower,        more preferably of 30 nM or lower, even more preferably of 20 nM        or lower, most preferably of 10 nM or lower, such as 5 nM, in an        Alphascreen assay as e.g., described in the experimental part        (see e.g., Example 1.5), and wherein the polypeptides comprise        only one human HGF binding immunoglobulin single variable domain        unit, and wherein full displacement means an average HGF        displacement of about 60% to 80% and more, preferably 95% or        more (e.g., when measured in an Alphascreen assay;

and/or:

-   -   fully displace human HGF (SEQ ID NO: 1) from human c-Met at an        average IC50 value of 50 nM or less, more preferably at an        average IC50 value of 30 nM or less, even more preferably at an        average IC50 value of 20 nM or less in an assay as e.g.        described in the experimental part (e.g., Example 1.6), and        wherein the polypeptides comprise only one human HGF binding        immunoglobulin single variable domain unit, and wherein full        displacement means an average HGF displacement of about 60% to        80% and more, preferably 95% or more (e.g. when measured        according to the ligand displacement assay in Example 1.6);

and/or such that they:

-   -   bind human HGF (SEQ ID NO: 1) with an average Kd value of 50 nM        or less, more preferably at an average Kd value of 30 nM or        less, even more preferably at an average Kd value of 20 nM or        less, such as less than 10, 9, 8, 7, 6, 5, 4, 3, 2 nM or even        less, such as less than 1 nM, or most preferably even less than        0.1 nM.

It should be appreciated that binding of the immunoglobulin singlevariable domains and/or polypeptides of the invention to (human) HGF mayresult in displacing (human) HGF from (human) c-Met as described herein.It should further be appreciated that binding of the immunoglobulinsingle variable domains and/or polypeptides of the invention to (human)HGF may result in inhibiting binding of (human) HGF to its cognatereceptor, such as, (human) c-Met as described herein.

Some preferred technical values for binding, displacing, migration orother in vivo and/or in vitro potency of the immunoglobulin singlevariable domains or polypeptides of the invention to HGF and inparticular human HGF (SEQ ID NO: 1) will become clear from the furtherdescription and examples herein.

For binding to HGF and in particular human HGF (SEQ ID NO: 1), an aminoacid sequence of the invention, such as an ISVD of the invention or apolypeptide of the invention, will usually contain within its amino acidsequence one or more amino acid residues or one or more stretches ofamino acid residues (i.e., with each “stretch” comprising two or aminoacid residues that are adjacent to each other or in close proximity toeach other, i.e., in the primary or tertiary structure of the amino acidsequence) via which the amino acid sequence of the invention can bind toHGF and in particular human HGF (SEQ ID NO: 1), which amino acidresidues or stretches of amino acid residues thus form the “site” forbinding to HGF and in particular human HGF (SEQ ID NO: 1) (also referredto herein as the “antigen binding site”).

The immunoglobulin single variable domains provided by the invention arepreferably in essentially isolated form (as defined herein), or formpart of a protein or polypeptide of the invention (as defined herein),which may comprise or essentially consist of one or more immunoglobulinsingle variable domains of the invention and which may optionallyfurther comprise one or more further immunoglobulin single variabledomains (all optionally linked via one or more suitable linkers). Forexample, and without limitation, a preferred aspect of the inventionprovides a polypeptide consisting essentially of one immunoglobulinsingle variable domain directed against human HGF and an immunoglobulinsingle variable domain directed against human serum albumin linked by apeptide linker (as defined herein), so as to provide a bispecificpolypeptide of the invention, respectively, and/or an immunoglobulinsingle variable domain directed against human EGFR also linked by apeptide linker (as defined herein), so as to provide a furtherbispecific or a trispecific polypeptide of the invention, all asdescribed herein. Such a protein or polypeptide may also be inessentially isolated form (as defined herein).

The immunoglobulin single variable domains and polypeptides of theinvention as such preferably essentially consist of a single amino acidchain that is not linked via disulphide bridges to any other amino acidsequence or chain (but that may or may not contain one or moreintramolecular disulphide bridges. For example, it is known that agentof the invention—as described herein—may sometimes contain a disulphidebridge between CDR3 and CDR1 or FR2). However, it should be noted thatone or more immunoglobulin single variable domains of the invention maybe linked to each other and/or to other immunoglobulin single variabledomains (e.g., via disulphide bridges) to provide peptide constructsthat may also be useful in the invention (for example Fab′ fragments,F(ab′)₂ fragments, ScFv constructs, “diabodies” and other multispecificconstructs. Reference is for example made to the review by Holliger andHudson, Nat Biotechnol. 2005; 23:1126-36 (incorporated by reference).

Generally, when an amino acid sequence of the invention (or a compound,construct or polypeptide comprising the same) is intended foradministration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is preferably either anamino acid sequence that does not occur naturally in said subject; or,when it does occur naturally in said subject, is in essentially isolatedform (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use,the immunoglobulin single variable domains of the invention (as well ascompounds, constructs and polypeptides comprising the same) arepreferably directed against human HGF and in particular human HGF withSEQ ID NO: 1; whereas for veterinary purposes, the immunoglobulin singlevariable domains and polypeptides of the invention are preferablydirected against HGF from the species to be treated, or at leastcross-reactive with HGF from the species to be treated.

Furthermore, an amino acid sequence of the invention may optionally, andin addition to the at least one binding site for binding against HGF andin particular human HGF (SEQ ID NO: 1), contain one or more furtherbinding sites for binding against other antigens, proteins or targets.

The efficacy of the immunoglobulin single variable domains andpolypeptides of the invention, and of compositions comprising the same,can be tested using any suitable in vitro assay, cell-based assay, invivo assay and/or animal model known per se, or any combination thereof,depending on the specific disease or disorder involved. Suitable assaysand animal models will be clear to the skilled person, and for exampleinclude ligand displacement assays (Burgess et al., Cancer Res 200666:1721-9), dimerization assays (WO2009/007427A2, Goetsch, 2009),signaling assays (Burgess et al., Mol Cancer Ther 9:400-9),proliferation/survival assays (Pacchiana et al., J Biol Chem 2010September M110.134031), cell adhesion assays (Holt et al., Haematologica2005 90:479-88) and migration assays (Kong-Beltran et al., Cancer Cell6:75-84), endothelial cell sprouting assays (Wang et al., J Immunol.2009; 183:3204-11), and in vivo xenograft models (Jin et al., CancerRes. 2008 68:4360-8), as well as the assays and animal models used inthe experimental part below and in the prior art cited herein.

Also, according to the invention, immunoglobulin single variable domainsand polypeptides that are directed against HGF from a first species ofwarm-blooded animal may or may not show cross-reactivity with HGF fromone or more other species of warm-blooded animal. For example,immunoglobulin single variable domains and polypeptides directed againsthuman HGF and in particular human HGF with SEQ ID NO: 1 may or may notshow cross reactivity with HGF from one or more other species ofprimates (such as, without limitation, monkeys from the genus Macaca(such as, and in particular, cynomolgus monkeys (Macaca fascicularis)and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ursinus))and/or with HGF from one or more species of animals that are often usedin animal models for diseases (for example mouse, rat, rabbit, pig ordog), and in particular in animal models for diseases and disordersassociated with HGF and in particular human HGF (SEQ ID NO: 1) (such asthe species and animal models mentioned herein). In this respect, itwill be clear to the skilled person that such cross-reactivity, whenpresent, may have advantages from a drug development point of view,since it allows the immunoglobulin single variable domains andpolypeptides against human HGF and in particular human HGF (SEQ IDNO: 1) to be tested in such disease models.

More generally, immunoglobulin single variable domains and polypeptidesof the invention that are cross-reactive with HGF from multiple speciesof mammal will usually be advantageous for use in veterinaryapplications, since it will allow the same amino acid sequence orpolypeptide to be used across multiple species. Thus, it is alsoencompassed within the scope of the invention that immunoglobulin singlevariable domains and polypeptides directed against HGF from one speciesof animal (such as immunoglobulin single variable domains andpolypeptides against human HGF (SEQ ID NO: 1)) can be used in thetreatment of another species of animal, as long as the use of theimmunoglobulin single variable domains and/or polypeptides provide thedesired effects in the species to be treated.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain, subunit or confirmation (where applicable) of HGF and inparticular human HGF (SEQ ID NO: 1) against which the immunoglobulinsingle variable domains and polypeptides of the invention are directed.For example, the immunoglobulin single variable domains and polypeptidesmay or may not be directed against the HGF/c-Met interaction site, andare as further defined herein.

Furthermore, immunoglobulin single variable domains with dualspecificity to HGF and c-Met are within the scope of this invention, aswell as with dual specificity to HGF and RON, and in particular to humanRON (Ming-Hai Wang et al., Acta Pharmacologica Sinica (2010) 31:1181-1188) are within the scope of this invention.

As further described herein, a polypeptide of the invention may containtwo or more immunoglobulin single variable domains of the invention thatare directed against HGF and in particular human HGF (SEQ ID NO: 1).Generally, such polypeptides will bind to HGF and in particular humanHGF (SEQ ID NO: 1) with increased avidity compared to a single aminoacid sequence of the invention. Such a polypeptide may for examplecomprise two immunoglobulin single variable domains of the inventionthat are directed against the same antigenic determinant, epitope, part,domain, subunit or confirmation (where applicable) of HGF and inparticular human HGF (SEQ ID NO: 1) (which may or may not be aninteraction site); or comprise at least one “first” amino acid sequenceof the invention that is directed against a first antigenic determinant,epitope, part, domain, subunit or conformation (where applicable) of HGFand in particular human HGF (SEQ ID NO: 1) (which may or may not be aninteraction site); and at least one “second” amino acid sequence of theinvention that is directed against a second antigenic determinant,epitope, part, domain, subunit or conformation (where applicable)different from the first (and which again may or may not be aninteraction site). Preferably, in such “biparatopic” polypeptides of theinvention, at least one amino acid sequence of the invention is directedagainst an interaction site (as defined herein), although the inventionin its broadest sense is not limited thereto. For instance, polypeptidesof the invention may be formatted e.g., in a biparatopic way such as tocombine monovalent building blocks directed against different epitopesas characterized in the experimental part.

Also, when the target is part of a binding pair (for example, areceptor-ligand binding pair), the immunoglobulin single variabledomains and polypeptides may be such that they compete with the cognatebinding partners, e.g., HGF for binding to c-Met, and/or such that they(fully or partially) neutralize binding of the binding partner to thetarget.

It is also expected that the immunoglobulin single variable domains andpolypeptides of the invention will generally bind to all naturallyoccurring or synthetic analogs, variants, mutants, alleles, parts andfragments of HGF and in particular human HGF (SEQ ID NO: 1); or at leastto those analogs, variants, mutants, alleles, parts and fragments of HGFand in particular human HGF (SEQ ID NO: 1) that contain one or moreantigenic determinants or epitopes that are essentially the same as theantigenic determinant(s) or epitope(s) to which the immunoglobulinsingle variable domains and polypeptides of the invention bind to HGFand in particular to human HGF (SEQ ID NO: 1). Again, in such a case,the immunoglobulin single variable domains and polypeptides of theinvention may bind to such analogs, variants, mutants, alleles, partsand fragments with an affinity and/or specificity that are the same as,or that are different from (i.e., higher than or lower than), theaffinity and specificity with which the immunoglobulin single variabledomains of the invention bind to (wild-type) HGF.

As HGF and in particular human HGF (SEQ ID NO: 1) exists in a monomericform and in one or more multimeric forms, e.g. in homodimeric form, itis within the scope of the invention that the immunoglobulin singlevariable domains and polypeptides of the invention i) only bind to HGFand in particular human HGF (SEQ ID NO: 1) in monomeric form, ii) onlybind to HGF and in particular human HGF (SEQ ID NO: 1) inmultimeric/dimeric (homo- and/or heterodimeric) form, or iii) bind toboth the monomeric and the multimeric form. In a preferred aspect of theinvention, the polypeptides of the invention prevent formation ofhomodimeric human HGF complexes. In another preferred aspect of theinvention, the polypeptides of the invention do not induce (even athigher concentration such as 10 nM or more, 50 nM or more, 100 nM ormore, or 500 nM or more) formation of homodimeric human HGF complexes.Again, in such a case, the polypeptides of the invention may bind to themonomeric form with an affinity and/or specificity that are the same as,or that are different from (i.e., higher than or lower than), theaffinity and specificity with which the immunoglobulin single variabledomains of the invention bind to the multimeric form.

Also, when HGF and in particular human HGF (SEQ ID NO: 1) can associatewith other proteins or polypeptides to form protein complexes (e.g.,with c-Met, but also with other receptors such as EGFR, HER3, plexins,integrins, CD44, RON), it is within the scope of the invention that theimmunoglobulin single variable domains and polypeptides of the inventionbind to HGF and in particular human HGF (SEQ ID NO: 1) in itsnon-associated state (and e.g., prevent ligand binding and/or preventsignalling), bind to HGF and in particular human HGF (SEQ ID NO: 1) inits associated state, or bind to both (preferably to the non-associatedstate). In all these cases, the immunoglobulin single variable domainsand polypeptides of the invention may bind to such associated proteincomplexes with an affinity and/or specificity that may be the same as ordifferent from (i.e., higher than or lower than) the affinity and/orspecificity with which the immunoglobulin single variable domains andpolypeptides of the invention bind to HGF and in particular human HGF(SEQ ID NO: 1) in its non-associated state.

Also, as will be clear to the skilled person, proteins or polypeptidesthat contain two or more immunoglobulin single variable domains directedagainst HGF and in particular human HGF (SEQ ID NO: 1), e.g.,“biparatopic” polypeptides of the invention, may bind with higheravidity to HGF and in particular human HGF (SEQ ID NO: 1) than thecorresponding monomeric amino acid sequence(s). For example, and withoutlimitation, proteins or polypeptides that contain two or moreimmunoglobulin single variable domains directed against differentepitopes of HGF and in particular human HGF (SEQ ID NO: 1) may (andusually will) bind with higher avidity than each of the differentmonomers, and proteins or polypeptides that contain two or moreimmunoglobulin single variable domains directed against HGF and inparticular human HGF (SEQ ID NO: 1) may (and usually will) bind alsowith higher avidity to a multimer (e.g., homodimer) of HGF and inparticular to a multimer (e.g., homodimer) of human HGF (SEQ ID NO: 1).

Generally, immunoglobulin single variable domains and polypeptides ofthe invention will at least bind to those forms of HGF and in particularhuman HGF (SEQ ID NO: 1) (including monomeric, multimeric, associatedand different conformational forms) that are the most relevant from abiological and/or therapeutic point of view, as will be clear to theskilled person.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of theimmunoglobulin single variable domains and polypeptides of theinvention, and/or to use proteins or polypeptides comprising oressentially consisting of one or more of such parts, fragments, analogs,mutants, variants, alleles and/or derivatives, as long as these aresuitable for the uses envisaged herein. Such parts, fragments, analogs,mutants, variants, alleles and/or derivatives will usually contain (atleast part of) a functional antigen-binding site for binding against HGFand in particular human HGF (SEQ ID NO: 1); and more preferably will becapable of specific binding to HGF and in particular human HGF (SEQ IDNO: 1), and even more preferably capable of binding to HGF and inparticular human HGF (SEQ ID NO: 1) with an EC50 value, average Ki, IC₅₀value concerning binding, migration, displacing and/or proliferationblocking and/or other measures for potency, as further described herein,(e.g., in the experimental part) that is as defined herein and suchparts, fragments, analogs, mutants, variants, alleles and/or derivativesmay be more potent, more stable, more soluble and may have the sameepitope. Some non-limiting examples of such parts, fragments, analogs,mutants, variants, alleles, derivatives, proteins and/or polypeptideswill become clear from the further description herein. Additionalfragments or polypeptides of the invention may also be provided bysuitably combining (i.e., by linking or genetic fusion) one or more(smaller) parts or fragments as described herein.

For a general description of immunoglobulin single variable domains,reference is made to the further description below, as well as to theprior art cited herein. In this respect, it should however be noted thatthis description and the prior art mainly describes immunoglobulinsingle variable domains of the so-called “V_(H)3 class” (i.e.,immunoglobulin single variable domains with a high degree of sequencehomology to human germline sequences of the V_(H)3 class such as DP-47,DP-51 or DP-29), which form a preferred aspect of this invention. Itshould, however, be noted that the invention in its broadest sensegenerally covers any type of immunoglobulin single variable domainsdirected against HGF and in particular human HGF (SEQ ID NO: 1), and forexample also covers the immunoglobulin single variable domains belongingto the so-called “V_(H)4 class” (i.e., immunoglobulin single variabledomains with a high degree of sequence homology to human germlinesequences of the V_(H)4 class such as DP-78), as for example describedin WO 07/118,670.

Generally, immunoglobulin single variable domains (in particular V_(HH)sequences and sequence optimized immunoglobulin single variable domains)can in particular be characterized by the presence of one or more“Hallmark residues” (as described herein) in one or more of theframework sequences (again as further described herein).

Thus, generally, an immunoglobulin single variable domain can be definedas an amino acid sequence with the (general) structure (cf. formula 1below)

FR1CDR1-FR2-CDR2-FR3CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively.

In a preferred aspect, the invention provides polypeptides comprising atleast an immunoglobulin single variable domain that is an amino acidsequence with the (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) at least one of the amino acid residues at positions 11, 37, 44,    45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering    are chosen from the Hallmark residues mentioned in Table A-1 below;    and in which:-   ii) said amino acid sequence has at least 80%, more preferably 90%,    even more preferably 95% amino acid identity with at least one of    the immunoglobulin single variable domains as shown in WO    2009/138519 (see SEQ ID NOs: 1 to 125 in WO 2009/138519), in which    for the purposes of determining the degree of amino acid identity,    the amino acid residues that form the CDR sequences (indicated with    X in the sequences) are disregarded; and in which:-   iii) the CDR sequences are generally as further defined herein    (e.g., the CDR1, CDR2 and CDR3 in a combination as provided in Table    (3-2), note that the CDR definitions are calculated according to the    Kabat numbering system).

TABLE A-1 Hallmark Residues in VHHs Position Human V_(H)3 HallmarkResidues  11 L, V; predominantly L L, S, V, M, W, F, T, Q, E, A, R, G,K, Y, N, P, I; preferably L  37 V, I, F; usually V F⁽¹⁾, Y, V, L, A, H,S, I, W, C, N, G, D, T, P, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G E⁽³⁾, Q⁽³⁾,G⁽²⁾, D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G⁽²⁾, E⁽³⁾ orQ⁽³⁾; most preferably G⁽²⁾ or Q⁽³⁾.  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, P, H, F, G, Q,S, E, T, Y, C, I, D, V; preferably L⁽²⁾ or R⁽³⁾  47⁽⁸⁾ W, Y F⁽¹⁾, L⁽¹⁾or W⁽²⁾ G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferablyW⁽²⁾, L⁽¹⁾ or F⁽¹⁾  83 R or K; usually R R, K⁽⁵⁾, T, E⁽⁵⁾, Q, N, S, I,V, G, M, L, A, D, Y, H; preferably K or R; most preferably K  84 A, T,D; predominantly A P⁽⁵⁾, S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E;preferably P 103 W W⁽⁴⁾, R⁽⁶⁾, G, S, K, A, M, Y, L, F, T, N, V, Q, P⁽⁶⁾,E, C; preferably W 104 G G, A, S, T, D, P, N, E, C, L; preferably G 108L, M or T; predominantly L Q, L⁽⁷⁾, R, P, E, K, S, T, M, A, H;preferably Q or L⁽⁷⁾ Notes: ⁽¹⁾In particular, but not exclusively, incombination with KERE or KQRE at positions 43-46. ⁽²⁾Usually as GLEW atpositions 44-47. ⁽³⁾Usually as KERE or KQRE at positions 43-46, e.g. asKEREL, KEREF, KQREL, KQREF, KEREG, KQREW or KQREG at positions 43-47.Alternatively, also sequences such as TERE (for example TEREL), TQRE(for example TQREL), KECE (for example KECEL or KECER), KQCE (forexample KQCEL), RERE (for example REREG), RQRE (for example RQREL, RQREFor RQREW), QERE (for example QEREG), QQRE, (for example QQREW, QQREL orQQREF), KGRE (for example KGREG), KDRE (for example KDREV) are possible.Some other possible, but less preferred sequences include for exampleDECKL and NVCEL. ⁽⁴⁾With both GLEW at positions 44-47 and KERE or KQREat positions 43-46. ⁽⁵⁾Often as KP or EP at positions 83-84 of naturallyoccurring V_(HH) domains. ⁽⁶⁾In particular, but not exclusively, incombination with GLEW at positions 44-47. ⁽⁷⁾With the proviso that whenpositions 44-47 are GLEW, position 108 is always Q in (non-humanized)V_(HH) sequences that also contain a W at 103. ⁽⁸⁾The GLEW group alsocontains GLEW-like sequences at positions 44-47, such as for exampleGVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER andELEW.

Again, such immunoglobulin single variable domains may be derived in anysuitable manner and from any suitable source, and may for example benaturally occurring V_(HH) sequences (i.e., from a suitable species ofCamelid, e.g., llama) or synthetic or semi-synthetic VHs or VLs (e.g.,from human). Such immunoglobulin single variable domains may include“humanized” or otherwise “sequence optimized” VHHs, “camelized”immunoglobulin sequences (and in particular camelized heavy chainvariable domain sequences, i.e., camelized VHs), as well as human VHs,human VLs, camelid VHHs that have been altered by techniques such asaffinity maturation (for example, starting from synthetic, random ornaturally occurring immunoglobulin sequences), CDR grafting, veneering,combining fragments derived from different immunoglobulin sequences, PCRassembly using overlapping primers, and similar techniques forengineering immunoglobulin sequences well known to the skilled person;or any suitable combination of any of the foregoing as further describedherein. As mentioned herein, a particularly preferred class ofimmunoglobulin single variable domains of the invention comprisesimmunoglobulin single variable domains with an amino acid sequence thatcorresponds to the amino acid sequence of a naturally occurring V_(HH)domain, but that has been “humanized”, i.e. by replacing one or moreamino acid residues in the amino acid sequence of said naturallyoccurring V_(HH) sequence (and in particular in the framework sequences)by one or more of the amino acid residues that occur at thecorresponding position(s) in a V_(H) domain from a conventional 4-chainantibody from a human being (e.g. indicated above). This can beperformed in a manner known per se, which will be clear to the skilledperson, for example on the basis of the further description herein andthe prior art on humanization referred to herein. Again, it should benoted that such humanized immunoglobulin single variable domains of theinvention can be obtained in any suitable manner known per se and thusare not strictly limited to polypeptides that have been obtained using apolypeptide that comprises a naturally occurring V_(HH) domain as astarting material.

Another particularly preferred class of immunoglobulin single variabledomains of the invention comprises immunoglobulin single variabledomains with an amino acid sequence that corresponds to the amino acidsequence of a naturally occurring V_(H) domain, but that has been“camelized”, i.e. by replacing one or more amino acid residues in theamino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe description herein. Such “camelizing” substitutions are preferablyinserted at amino acid positions that form and/or are present at theV_(H)-V_(L) interface, and/or at the so-called Camelidae hallmarkresidues, as defined herein (see also for example WO 94/04678 and Daviesand Riechmann (1994 and 1996)). Preferably, the V_(H) sequence that isused as a starting material or starting point for generating ordesigning the camelized immunoglobulin single variable domains ispreferably a V_(H) sequence from a mammal, more preferably the V_(H)sequence of a human being, such as a V_(H)3 sequence. However, it shouldbe noted that such camelized immunoglobulin single variable domains ofthe invention can be obtained in any suitable manner known per se andthus are not strictly limited to polypeptides that have been obtainedusing a polypeptide that comprises a naturally occurring V_(H) domain asa starting material.

For example, again as further described herein, both “humanization” and“camelization” can be performed by providing a nucleotide sequence thatencodes a naturally occurring V_(HH) domain or V_(H) domain,respectively, and then changing, in a manner known per se, one or morecodons in said nucleotide sequence in such a way that the new nucleotidesequence encodes a “humanized” or “camelized” immunoglobulin singlevariable domains of the invention, respectively. This nucleic acid canthen be expressed in a manner known per se, so as to provide the desiredimmunoglobulin single variable domains of the invention. Alternatively,based on the amino acid sequence of a naturally occurring V_(HH) domainor V_(H) domain, respectively, the amino acid sequence of the desiredhumanized or camelized immunoglobulin single variable domains of theinvention, respectively, can be designed and then synthesized de novousing techniques for peptide synthesis known per se. Also, based on theamino acid sequence or nucleotide sequence of a naturally occurringV_(HH) domain or V_(H) domain, respectively, a nucleotide sequenceencoding the desired humanized or camelized immunoglobulin singlevariable domains of the invention, respectively, can be designed andthen synthesized de novo using techniques for nucleic acid synthesisknown per se, after which the nucleic acid thus obtained can beexpressed in a manner known per se, so as to provide the desiredimmunoglobulin single variable domains of the invention.

In a further preferred aspect, the invention provides polypeptidescomprising one immunoglobulin single variable domain with amino acidsequence selected from the group consisting of amino acid sequences withSEQ ID NOs: 6 to 27, preferably SEQ ID NOs: 7, 8, 10, 15, 16, 18 and20-25, even more preferably SEQ ID NOs: 7 and 18, (see experimentalpart) and one immunoglobulin single variable domain with amino acidsequence selected from the group consisting of moieties providing anincreased half-life (see below).

In a further preferred aspect, the invention provides polypeptidescomprising at least an immunoglobulin single variable domain with aminoacid sequence selected from the group consisting of amino acid sequencesthat essentially consist of 4 framework regions (FR1 to FR4,respectively) and 3 complementarity determining regions (CDR1 to CDR3,respectively), in which the CDR sequences of said amino acid sequenceshave at least 70% amino acid identity, preferably at least 80% aminoacid identity, more preferably at least 90% amino acid identity, such as95% amino acid identity or more or even essentially 100% amino acididentity with the CDR sequences (see Table B-2) of at least one of theimmunoglobulin single variable domains of SEQ ID NOs: 6 to 27,preferably SEQ ID NOs: 7, 8, 10, 15, 16, 18 and 20-25, even morepreferably SEQ ID NOs: 7 and 18 (see experimental part). This degree ofamino acid identity can for example be determined by determining thedegree of amino acid identity (in a manner described herein) betweensaid amino acid sequence and one or more of the sequences of SEQ ID NOs:6 to 27, preferably SEQ ID NOs: 7, 8, 10, 15, 16, 18 and 20-25, evenmore preferably SEQ ID NOs: 7 and 18 (see experimental part), in whichthe amino acid residues that form the framework regions are disregarded.Such polypeptides and/or immunoglobulin single variable domains of theinvention may further provide the following:

-   (i) polypeptides comprising at least one (preferably one)    immunoglobulin single variable domain that is directed against (as    defined herein) HGF and in particular human HGF (SEQ ID NO: 1) and    that has at least 80%, preferably at least 85%, such as 90% or 95%    or more sequence identity with at least one of the immunoglobulin    single variable domains of SEQ ID NOs: 6 to 27, preferably SEQ ID    NOs: 7, 8, 10, 15, 16, 18 and 20-25, even more preferably SEQ ID    NOs: 7 and 18 (see experimental part); and/or-   (ii) polypeptides comprising at least one (preferably one)    immunoglobulin single variable domain that is directed against (as    defined herein) HGF and in particular human HGF (SEQ ID NO: 1) and    that cross-block (as defined herein) the binding of at least one of    the immunoglobulin single variable domains of SEQ ID NOs: 6 to 27,    preferably SEQ ID NOs: 7, 8, 10, 15, 16, 18 and 20-25, even more    preferably SEQ ID NOs: 7 and 18 (see experimental part) to HGF and    in particular human HGF (SEQ ID NO: 1) and/or that compete with at    least one of the immunoglobulin single variable domains of SEQ ID    NOs: 6 to 27, preferably SEQ ID NOs: 7, 8, 10, 15, 16, 18 and 20-25,    even more preferably SEQ ID NOs: 7 and 18 (see experimental part)    for binding to HGF and in particular human HGF (SEQ ID NO: 1), and    of which immunoglobulin single variable domains may be as further    described herein; and/or-   (iii) polypeptides of the invention that comprise one or more    (preferably one) of such immunoglobulin single variable domains    (which may be as further described herein, and may for example be    bispecific (e.g. also bind to serum albumin) and/or biparatopic    polypeptides as described herein), and nucleic acid sequences that    encode such immunoglobulin single variable domains and polypeptides.    Such immunoglobulin single variable domains and polypeptides do not    include any naturally occurring ligands.

The polypeptides of the invention comprise or essentially consist of atleast one immunoglobulin single variable domain of the invention. Somepreferred, but non-limiting examples of immunoglobulin single variabledomains of the invention are given in SEQ ID NOs: 6 to 27, preferablySEQ ID NOs: 7, 8, 10, 15, 16, 18 and 20-25, even more preferably SEQ IDNOs: 7 and 18 (see experimental part).

EGFR consists of an extracellular ligand-binding domain, a transmembranedomain and an intracellular tyrosine kinase domain (Yarden et al. 2001,Nature Rev. Mol. Cell Biol. 2:127-137). Aberrant activation of EGFRmediated signalling has been implicated in processes involved in tumorgrowth and progression, including tumor cell proliferation,angiogenesis, metastasis, inhibition of apoptosis and resistance toradio- or chemotherapy (Grünwald, Hidalgo 2003 J. Natl. Cancer Inst.95:851-867; and references therein). EGFR is expressed in a wide varietyof tumors of epithelial origin, including >40% of NSCLC(non-small-cell-lung cancer), >95% of head and neck cancer, >30% ofpancreatic cancer, >90% of renal carcinoma, >35% of ovarian cancer, >40%of glioma and >31% of bladder cancer (Salomon et al. 1995. Crit. ReviewOncol. Hematol, 19:183-232). Since high levels of EGFR expression arecorrelated to disease progression, increased metastasis and poorprognosis, this provides a strong rationale for developing effectiveEGFR targeting antibodies for the treatment of various solid tumors.

Identification of mAbs inhibiting EGFR is an approach used in clinicaldevelopment to target aberrant signalling of EGFR in malignantneoplasia. Examples of such EGFR targeting antibodies are IMC-C225(Erbitux, Imclone), EMD72000 (Merck Darmstadt), ABX-EGF (Abgenix), h-R3(theraCIM, YM Biosciences) and Humax-EGFR (Genmab). The mechanism ofaction of these antibodies relies on the inhibition with ligand bindingto the receptor and subsequent inhibition of receptortransphosphorylation and the downstream signaling cascade. Mab 225 (ofwhich Erbitux is the chimeric derivative), the 225-derived F(ab′)₂fragment are able to induce EGFR internalization and modest receptorsequestration but only after sustained incubation with EGFR expressingcells. The monovalent 225-derived Fab′ fragment however only inducesreceptor downregulation after preincubation with a rabbit anti-mouseantibody (Fan et al., 1993 J. Biol. Chem. 268:21073-21079; Fan et al.,1994 J. Biol. Chem. 269:27595-27602). These antibodies show anantitumoral activity against a broad panel of human tumor xenografts(reviewed in Grünwald & Hidalgo 2003 J. Natl. Cancer Inst. 95:851-867).

However, the known antibody-based therapeutics binding to the EGFreceptor are cytostatic instead of cytotoxic. Indeed none of theseantibodies or the presently available small molecule drugs is completelyeffective for the treatment of cancer. Moreover, for some patientstherapeutic application of EGFR inhibitors is limited by serioustoxicity.

WO 05/044858, WO 04/041867 and WO07/042,289 already describe anti-EGFRNanobodies and polypeptides with improved properties over standardantibodies. What is more, biodistribution of αEGFR-αEGFR-αAlb (50 kDa)was comparable to cetuximab (150 kDa), while it showed faster and deepertumor penetration. The latter indicates that Nanobodies might havedistinguished potential in comparison to conventional mAbs for use incancer treatment.

In addition, multispecific constructs comprising the polypeptides of thepresent invention have improved efficacy in modulating signalling over acombination of the individual polypeptides of the present invention. Inparticular, a multispecific construct comprising (a) one or morepolypeptides modulating HGF-mediated signalling as described herein, and(b) one or more polypeptides modulating EGFR-mediated signalling isexceptionally useful in the diagnosis, prevention and treatment ofdiseases and disorders as set out above. The multispecific construct isparticular useful in the diagnosis, prevention and treatment of cancer,in particular of non-small cell lung cancer.

The polypeptides and Nanobodies described in WO 05/044858, WO 04/041867,and/or WO07/042,289 are particularly preferred as polypeptidesmodulating EGFR-mediated signalling in the multispecific constructs ofthe present invention. Accordingly, the present invention relates to amultispecific, such as, for instance, a bispecific or trispecific (oreven tetraspecific), construct comprising at least one ISVD against EGFRand at least one ISVD against HGF, and optionally against VEGF. In sucha multispecific, e.g. bispecific or trispecific (or even tetraspecific),polypeptide construct, the Nanobodies and polypeptides against HGFdescribed herein can be combined with one or more of the anti-EGFRNanobodies and polypeptides described in WO 05/044858, WO 04/041867, andWO07/042,289 (all of which are specifically incorporated in its entiretyherein).

Hence, the present invention relates to a multispecific construct of (a)one or more polypeptides modulating HGF-mediated signalling and (b) oneor more polypeptides modulating EGFR-mediated signalling, in particularEGFR-mediated signalling (c) and possibly Alb-Nanobodies, for use in thediagnosis, prevention and treatment of diseases and disorders as set outabove, in particular non-small cell lung cancer.

Development of a vascular system is a fundamental requirement for manyphysiological and pathological processes. It is now well establishedthat angiogenesis is implicated in the pathogenesis of a variety ofdisorders, including solid tumors and metastasis. In the case of tumorgrowth, angiogenesis appears to be crucial for the transition fromhyperplasia to neoplasia, and for providing nourishment for the growthand metastasis of the tumor. Folkman et al., Nature 339:58 (1989). Theprocess of vascular development is tightly regulated, in which vascularendothelial growth factor (VEGF) has been identified as the key factorinvolved in stimulating angiogenesis and in inducing vascularpermeability. Ferrara et al., Endocr. Rev. 18:4-25 (1997). The term“VEGF” or “VEGF-A” is used to refer to the 165-amino acid human vascularendothelial cell growth factor and related 121-, 189-, and 206-aminoacid human vascular endothelial cell growth factors, as described byLeung et al. Science, 246:1306 (1989), and Houck et al. Mol. Endocrin.,5:1806 (1991), together with the naturally occurring allelic andprocessed forms thereof. “VEGF biological activity” includes binding toany VEGF receptor or any VEGF signaling activity such as regulation ofboth normal and abnormal angiogenesis and vasculogenesis (Ferrara andDavis-Smyth (1997) Endocrine Rev. 18:4-25; Ferrara (1999) J. Mol. Med.77:527-543).

Most clinical experience has been obtained with A4.6.1, also calledbevacizumab (Avastin®; Genentech, San Francisco, Calif.). Avastin incombination with chemotherapy is, however, plagued by side-effects(hemorrhages, arterial thromboembolism, hypertension, gastrointestinal(GI) perforations, wound healing problems, proteinuria and congestiveheart failure) which are primarily due to the fact that the anti-VEGFactivity is not restricted to the site of the tumor, but persists incirculation over a long period of time. This results in a shift ofphysiological to pathophysiological activity of the peripheralendothelial cells. Anti-VEGF strategies using a recombinant humanizedanti-VEGF Fab (rhuFab VEGF, Ranibizumab or Lucentis™) for the treatmentof a chronic disease is, however, not ideal because of the risk ofendophthalmitis, vitreous hemorrhage, and retinal detachment.

WO 08/101,985 already describes anti-VEGF Nanobodies and polypeptideswith improved properties over standard antibodies.

However, the multispecific constructs comprising the polypeptides of thepresent invention have improved efficacy in modulating signalling over acombination of the individual polypeptides of the present invention. Inparticular, a multispecific construct comprising (a) one or morepolypeptides modulating HGF signalling as described herein, and (b) oneor more polypeptides modulating VEGF-mediated signalling, and optionallyEGFR-mediated signalling and possibly Alb-polypeptides, is exceptionallyuseful in the diagnosis, prevention and treatment of diseases anddisorders as set out above. The multispecific construct is particularuseful in the diagnosis, prevention and treatment of cancer, inparticular of non-small cell lung cancer.

The polypeptides and Nanobodies described in WO 08/101,985 areparticularly preferred as polypeptides modulating VEGF-mediatedsignalling in the multispecific constructs of the present invention.Accordingly, the present invention relates to a multispecific, such asfor instance a bispecific, trispecific, or tetraspecific constructcomprising at least one ISVD against HGF and at least one ISVD againstVEGF, and optionally against EGFR. In such a multispecific, e.g.bispecific, trispecific or tetraspecific, polypeptide construct, theNanobodies and polypeptides against HGF described herein can be combinedwith one or more of the anti-VEGF Nanobodies and polypeptides describedin WO 08/101,985 (which is specifically incorporated in its entiretyherein).

Hence, the present invention relates to a multispecific construct of (a)one or more polypeptides modulating HGF-mediated signalling and (b) oneor more polypeptides modulating VEGF-mediated signalling, in particularhuman VEGF-mediated signalling, and optionally (c) one or morepolypeptides modulating EGFR-mediated signalling, in particular humanEGFR-mediated signalling, for use in the diagnosis, prevention andtreatment of diseases and disorders as set out above, in particularnon-small cell lung cancer. In particular aspects, the present inventionprovides combination therapies for treating a pathological condition,such as cancer, wherein a HGF antagonist is combined with a VEGFantagonist, or wherein a HGF antagonist is combined with a VEGFantagonist and an EGFR antagonist, thereby providing significantanti-tumor activity.

Generally, proteins or polypeptides that comprise or essentially consistof a single immunoglobulin single variable domain will be referred toherein as “monovalent” proteins or polypeptides or as “monovalentconstructs”. Proteins and polypeptides that comprise or essentiallyconsist of two or more immunoglobulin single variable domains (such asat least two immunoglobulin single variable domains of the invention orat least one immunoglobulin single variable domain of the invention andat least one other immunoglobulin single variable domain) will bereferred to herein as “multivalent” proteins or polypeptides or as“multivalent constructs”, and these may provide certain advantagescompared to the corresponding monovalent immunoglobulin single variabledomains of the invention. Some non-limiting examples of such multivalentconstructs will become clear from the further description herein.

For example a “bivalent” polypeptide of the invention comprises twoISVDs, optionally linked via a linker sequence, whereas a “trivalent”polypeptide of the invention comprises three ISVDs, optionally linkedvia two linker sequences, whereas a “tetravalent” polypeptide of theinvention comprises four ISVDs, optionally linked via three linkersequences; etc.; in which at least one of the ISVDs present in thepolypeptide or construct, and up to all of the ISVDs present in thepolypeptide or construct, is/are an ISVD(s).

In a multivalent polypeptide of the invention the two or more ISVDs maybe the same or different, and may be directed against the same antigenor antigenic determinant (for example against the same part(s) orepitope(s) or against different parts or epitopes) or may alternativelybe directed against different antigens or antigenic determinants; or anysuitable combination thereof. For example, a bivalent polypeptide of theinvention may comprise (a) two identical ISVDs; (b) a first ISVDdirected against a first antigenic determinant of a protein or antigenand a second ISVD directed against the same antigenic determinant ofsaid protein or antigen which is different from the first ISVD; (c) afirst ISVD directed against a first antigenic determinant of a proteinor antigen and a second ISVD directed against another antigenicdeterminant of said protein or antigen; or (d) a first ISVD directedagainst a first protein or antigen and a second ISVD directed against asecond protein or antigen (i.e., different from said first antigen).Similarly, a trivalent polypeptide of the invention may, for example andwithout being limited thereto, comprise (a) three identical ISVDs; (b)two identical ISVDs against a first antigenic determinant of an antigenand a third ISVD directed against a different antigenic determinant ofthe same antigen; (c) two identical ISVDs against a first antigenicdeterminant of an antigen and a third ISVD directed against a secondantigen different from said first antigen; (d) a first ISVD directedagainst a first antigenic determinant of a first antigen, a second ISVDdirected against a second antigenic determinant of said first antigenand a third ISVD directed against a second antigen different from saidfirst antigen; or (e) a first ISVD directed against a first antigen, asecond ISVD directed against a second antigen different from said firstantigen, and a third ISVD directed against a third antigen differentfrom said first and second antigen. Similarly, a tetravalent polypeptideof the invention may, for example and without being limited thereto,comprise (a) four identical ISVDs; (b) three identical ISVDs against afirst antigenic determinant of a first antigen and one ISVD directedagainst a different antigenic determinant of the same antigen; (c) threeidentical ISVDs against a first antigenic determinant of a first antigenand one ISVD directed against a second antigen, different from saidfirst antigen; (d) two identical ISVDs against a first antigenicdeterminant of an antigen and two ISVDs directed against a differentantigenic determinant of the same antigen; (e) two identical ISVDsagainst a first antigenic determinant of an antigen, one ISVD directedagainst a different antigenic determinant of the same antigen, and oneISVDs directed against a second antigen different from said firstantigen; (f) two identical ISVDs against a first antigenic determinantof an antigen, two ISVDs directed against a second antigen, wherein saidsecond antigen is different from said first antigen; (g) two identicalISVDs against a first antigenic determinant of an antigen, one ISVDdirected against a second antigen, wherein said second antigen isdifferent from said first antigen, and one ISVD directed against a thirdantigen, wherein said third antigen is different from said first andsecond antigen; (h) a first ISVD directed against a first antigenicdeterminant of a first antigen, a second ISVD directed against a secondantigenic determinant of said first antigen, a third and a fourth ISVDdirected against a second antigen different from said first antigen; (i)a first ISVD directed against a first antigenic determinant of a firstantigen, a second ISVD directed against a second antigenic determinantof said first antigen, a third ISVD directed against a second antigendifferent from said first antigen and a fourth ISVD directed against athird antigen different from said first antigen and said second antigen;or (j) a first ISVD directed against a first antigen, a second ISVDdirected against a second antigen different from said first antigen, athird ISVD directed against a third antigen different from said firstand second antigen, and a fourth ISVD directed against a fourth antigendifferent from said first, said second and said third antigen.

Polypeptides of the invention that contain at least two ISVDs, in whichat least one ISVD is directed against a first antigen (i.e., againstHGF) and at least one ISVD is directed against a second antigen (i.e.,different from HGF, e.g. EGFR or VEGF), will also be referred to as“multispecific” polypeptides of the invention, and the ISVDs present insuch polypeptides will also be referred to herein as being in a“multivalent format”. Thus, for example, a “bispecific” polypeptide ofthe invention is a polypeptide that comprises at least one ISVD directedagainst a first antigen (i.e. HGF) and at least one further ISVDdirected against a second antigen (i.e., different from HGF, such as,for instance, EGFR or VEGF), whereas a “trispecific” polypeptide of theinvention is a polypeptide that comprises at least one ISVD directedagainst a first antigen (i.e., HGF), at least one further ISVD directedagainst a second antigen (i.e., different from HGF, such as for instanceEGFR or VEGF) and at least one further ISVD directed against a thirdantigen (i.e., different from both HGF and the second antigen, e.g.,EGFR or VEGF), whereas a “tetraspecific” polypeptide of the invention isa polypeptide that comprises at least one ISVD directed against a firstantigen (i.e., HGF), at least one further ISVD directed against a secondantigen (i.e., different from HGF, such as, for instance EGFR), at leastone further ISVD directed against a third antigen (i.e., different fromboth HGF and the second antigen EGFR, such as for instance VEGF), atleast one further ISVD directed against a fourth antigen (i.e.,different from the antigens HGF, EGFR as well as VEGF, such as, forinstance, serum albumin); etc.

Accordingly, in its simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first ISVD directed against HGF, and a second ISVDdirected against a second antigen, such as EGFR or VEGF, in which saidfirst and second ISVD may optionally be linked via a linker sequence (asdefined herein); whereas a trispecific polypeptide of the invention inits simplest form is a trivalent polypeptide of the invention (asdefined herein), comprising a first ISVD directed against HGF, a secondISVD directed against a second antigen, such as, for instance, EGFR orVEGF, and a third ISVD directed against a third antigen, different formHGF and said second antigen (e.g., EGFR or VEGF), in which said first,second and third ISVDs may optionally be linked via one or more, and inparticular one and more in particular two, linker sequences; whereas atetraspecific polypeptide of the invention in its simplest form is atetravalent polypeptide of the invention (as defined herein), comprisinga first ISVD directed against HGF, a second ISVD directed against asecond antigen, such as, for instance, EGFR, a third ISVD directedagainst a third antigen, such as VEGF, and a fourth ISVD directedagainst a fourth antigen different from HGF, EGFR and VEGF, in whichsaid first, second, third and fourth ISVDs may optionally be linked viaone or more, and in particular one or more in particular three, linkersequences.

However, as will be clear from the description, the invention is notlimited thereto, in the sense that a multispecific polypeptide of theinvention may comprise at least one ISVD against HGF and any number ofISVDs directed against one or more antigens different from HGF,respectively.

According to a specific, but non-limiting embodiment, a polypeptide asdescribed herein comprises at least one ISVD against HGF and at leastone ISVD against EGFR and/or VEGF, optionally linked using one or moresuitable linkers. In such a bispecific polypeptide construct, theNanobodies and polypeptides against HGF described herein can be combinedwith one or more of the anti-EGFR Nanobodies and polypeptides describedin WO 05/044858, WO 04/041867 and/or WO07/042,289, and/or with one ormore of the anti-VEGF Nanobodies and polypeptides described inWO08/101,985.

Bispecific polypeptides that comprise two binding moieties, such as forinstance two ISVDs, wherein each binding moiety is specific for a tumorassociated antigen (i.e., an antigen expressed on a tumor cell, alsocalled ‘tumor marker’), are highly advantageous in tumor targeting. Suchbispecific polypeptides are capable of simultaneously targeting twotumor associated antigens, resulting in enhanced tumor specificity. Itis known that most tumor markers are not truly tumor specific but alsooccur (mostly at lower levels) on normal tissues or cells. Monospecificbinding moieties, ISVDs or polypeptides against only one tumor markerwill therefore also recognize those normal tissues or cells resulting ina non-specific cell arrest or killing. Polypeptides that are specificfor two or more markers on one or more tumor cells will be much moretumor specific and provide a better specific binding. They can thusblock simultaneously multiple receptor activation and downstream signaltransduction pathways, and provide a better inhibition of tumorproliferation and arrest or killing of the tumor cells.

Accordingly, the present invention also relates to a bispecific ormultispecific polypeptide, comprising or essentially consisting of atleast two binding moieties, such as two ISVDs, wherein at least one ofsaid at least two binding moieties is directed against HGF, and theother binding moiety is directed against EGFR or VEGF. In a particularembodiment, said at least two binding moieties have a moderate or lowaffinity to their individual tumor associated antigen (such as, forinstance, HGF and EGFR or VEGF) and, accordingly, have only a reducedretention on normal tissues or cells expressing one of the tumorassociated antigens. Those at least two binding moieties, howeverpreferentially target (have a high avidity for) tumor cells that expressboth antigens (such as, for instance, HGF and EGFR or VEGF) recognizedby the bispecific or multispecific polypeptide.

Accordingly, the present invention also relates to a trispecific ormultispecific polypeptide, comprising or essentially consisting of atleast three binding moieties, such as three ISVDs, wherein at least oneof said at least three binding moieties is directed against HGF, onebinding moiety is directed against EGFR and one binding moiety isdirected against VEGF. In a particular embodiment, two of said at leastthree binding moieties have a moderate or low affinity to theirindividual tumor associated antigen (such as, for instance, HGF andEGFR) and, accordingly, have only a reduced retention on normal tissuesor cells expressing one of the tumor associated antigens. Those at leasttwo binding moieties, however preferentially target (have a high avidityfor) tumor cells that express both antigens (such as, for instance, HGFand EGFR) recognized by the bispecific, trispecific or multispecificpolypeptide.

EGFR, for example, is over-expressed on tumors in breast cancer, coloncancer, ovarian cancer, lung cancer and head and neck cancer.

By simultaneous targeting two of these tumor associated antigens, ordifferent epitopes on one of these tumor associated antigens, a muchmore selective and/or enhanced tumor targeting is obtained.

Therefore, in a preferred embodiment, the invention also provides abispecific or trispecific polypeptide comprising or essentiallyconsisting of a Nanobody directed against HGF and a Nanobody directedagainst EGFR and optionally against VEGF. The polypeptide of theinvention may comprise or essentially consist of a Nanobody directedagainst HGF and a Nanobody directed against EGFR. The polypeptide of theinvention may comprise or essentially consist of a Nanobody directedagainst HGF and a Nanobody directed against VEGF. Also, the polypeptideof the invention may comprise or essentially consist of a Nanobodydirected against HGF, a Nanobody directed against EGFR and a Nanobodydirected against VEGF.

Also encompassed within the scope of the present invention arebispecific or multispecific polypeptides comprising or essentiallyconsisting of at least two Nanobodies of which one of said at least twoNanobodies has a decreased or increased affinity for its antigen, uponbinding by the other Nanobodies to its antigen. Such binding is called‘conditional bispecific or multispecific binding’. Such bispecific ormultispecific polypeptide is also called ‘a conditionally bindingbispecific or multispecific polypeptide of the invention’.

Binding of the antigen by the first of said at least two Nanobodies maymodulate, such as enhance, reduce or inhibit, binding of the antigen bythe second of said at least two Nanobodies. In an embodiment, binding bythe first of said at least two Nanobodies stimulates binding by thesecond of said at least two Nanobodies. In another embodiment, bindingby the first of said at least two Nanobodies at least partially inhibitsbinding by the second of said at least two Nanobodies. In such anembodiment, the polypeptide of the invention may, for example, bemaintained in the body of a subject organism in vivo through binding toa protein which increases the half-life of the polypeptide until such atime as it becomes bound to its second target antigen and dissociatesfrom the half-life increasing protein.

Modulation of binding in the above context is achieved as a consequenceof the structural proximity of the antigen binding sites of theNanobodies relative to one another. Such structural proximity can beachieved by the nature of the structural components linking the two ormore antigen binding sites, e.g., by the provision of a linker with arelatively rigid structure that holds the antigen binding sites in closeproximity. Advantageously, the two or more antigen binding sites are inphysically close proximity to one another such that one site modulatesthe binding of the antigen at another site by a process which involvessteric hindrance and/or conformational changes within the polypeptide.

In another aspect, the invention relates to a compound or construct, andin particular to a protein or polypeptide (also referred to herein as a“compound of the invention” or “polypeptide of the invention”,respectively) that comprises or essentially consists of one or more(preferably one) immunoglobulin single variable domains directed tohuman HGF (or suitable fragments thereof), and optionally furthercomprises one or more other groups, residues, moieties or binding units.As will become clear to the skilled person from the further disclosureherein, such further groups, residues, moieties, binding units orimmunoglobulin single variable domains may or may not provide furtherfunctionality to the amino acid sequence of the invention (and/or to thecompound or construct in which it is present) and may or may not modifythe properties of the amino acid sequence of the invention.

As will be clear from the further description above and herein, thismeans that the immunoglobulin single variable domains of the inventioncan be used as “building blocks” to form polypeptides of the invention,i.e., by suitably combining them with other groups, residues, moietiesor binding units, in order to form compounds or constructs as describedherein (such as, without limitations, the biparatopic, triparatopic,tetraparatopic, bi/tri/tetra/multivalent and bi/tri/tetra/multispecificpolypeptides of the invention described herein) which combine within onemolecule one or more desired properties or biological functions.

The compounds or polypeptides of the invention can generally be preparedby a method which comprises at least one step of suitably linking theone or more immunoglobulin single variable domains of the invention tothe one or more further groups, residues, moieties or binding units,optionally via the one or more suitable linkers, so as to provide thecompound or polypeptide of the invention. Polypeptides of the inventioncan also be prepared by a method which generally comprises at least thesteps of providing a nucleic acid that encodes a polypeptide of theinvention, expressing said nucleic acid in a suitable manner, andrecovering the expressed polypeptide of the invention. Such methods canbe performed in a manner known per se, which will be clear to theskilled person, for example on the basis of the methods and techniquesfurther described herein.

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence ofthe invention, is also referred to herein as “formatting” said aminoacid sequence of the invention; and an amino acid of the invention thatis made part of a compound or polypeptide of the invention is said to be“formatted” or to be “in the format of” said compound or polypeptide ofthe invention. Examples of ways in which an amino acid sequence of theinvention can be formatted and examples of such formats will be clear tothe skilled person based on the disclosure herein; and such formattedimmunoglobulin single variable domains form a further aspect of theinvention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional immunoglobulin single variable domains,such that the compound or construct is a (fusion) protein or (fusion)polypeptide. In a preferred but non-limiting aspect, said one or moreother groups, residues, moieties or binding units are immunoglobulinsequences. Even more preferably, said one or more other groups,residues, moieties or binding units are chosen from the group consistingof domain antibodies, immunoglobulin single variable domains that aresuitable for use as a domain antibody, single domain antibodies,immunoglobulin single variable domains (ISVDs) that are suitable for useas a single domain antibody, “dAb”'s, immunoglobulin single variabledomains that are suitable for use as a dAb, or Nanobodies.Alternatively, such groups, residues, moieties or binding units may forexample be chemical groups, residues, moieties, which may or may not bythemselves be biologically and/or pharmacologically active. For example,and without limitation, such groups may be linked to the one or moreimmunoglobulin single variable domains of the invention so as to providea “derivative” of an amino acid sequence or polypeptide of theinvention, as further described herein.

Also within the scope of the present invention are compounds orconstructs, which comprise or essentially consist of one or morederivatives as described herein, and optionally further comprise one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are immunoglobulin singlevariable domains. In the compounds or constructs described above, theone or more immunoglobulin single variable domains of the invention andthe one or more groups, residues, moieties or binding units may belinked directly to each other and/or via one or more suitable linkers orspacers. For example, when the one or more groups, residues, moieties orbinding units are immunoglobulin single variable domains, the linkersmay also be immunoglobulin single variable domains, so that theresulting compound or construct is a fusion protein or fusionpolypeptide.

In a specific, but non-limiting aspect of the invention, which will befurther described herein, the polypeptides of the invention have anincreased half-life in serum (as further described herein) compared tothe immunoglobulin single variable domain from which they have beenderived. For example, an immunoglobulin single variable domain of theinvention may be linked (chemically or otherwise) to one or more groupsor moieties that extend the half-life (such as PEG), so as to provide aderivative of an amino acid sequence of the invention with increasedhalf-life.

In a specific aspect of the invention, a compound of the invention or apolypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such compounds and polypeptideswill become clear to the skilled person based on the further disclosureherein, and for example comprise immunoglobulin single variable domainsor polypeptides of the invention that have been chemically modified toincrease the half-life thereof (for example, by means of pegylation);immunoglobulin single variable domains of the invention that comprise atleast one additional binding site for binding to a serum protein (suchas serum albumin); or polypeptides of the invention which comprise atleast one amino acid sequence of the invention that is linked to atleast one moiety (and in particular at least one amino acid sequence)which increases the half-life of the amino acid sequence of theinvention. Examples of polypeptides of the invention which comprise suchhalf-life extending moieties or immunoglobulin single variable domainswill become clear to the skilled person based on the further disclosureherein; and for example include, without limitation, polypeptides inwhich the one or more immunoglobulin single variable domains of theinvention are suitably linked to one or more serum proteins or fragmentsthereof (such as (human) serum albumin or suitable fragments thereof) orto one or more binding units that can bind to serum proteins (such as,for example, domain antibodies, immunoglobulin single variable domainsthat are suitable for use as a domain antibody, single domainantibodies, immunoglobulin single variable domains that are suitable foruse as a single domain antibody, “dAb”'s, immunoglobulin single variabledomains that are suitable for use as a dAb, or Nanobodies that can bindto serum proteins such as serum albumin (such as human serum albumin),serum immunoglobulins such as IgG, or transferrin; reference is made tothe further description and references mentioned herein); polypeptidesin which an amino acid sequence of the invention is linked to an Fcportion (such as a human Fc) or a suitable part or fragment thereof; orpolypeptides in which the one or more immunoglobulin single variabledomains of the invention are suitable linked to one or more smallproteins or peptides that can bind to serum proteins, such as, withoutlimitation, the proteins and peptides described in WO 91/01743, WO01/45746, WO 02/076489, WO2008/068280, WO2009/127691 andPCT/EP2011/051559.

Generally, the compounds or polypeptides of the invention with increasedhalf-life preferably have a half-life that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence of the invention per se. For example,the compounds or polypeptides of the invention with increased half-lifemay have a half-life e.g., in humans that is increased with more than 1hours, preferably more than 2 hours, more preferably more than 6 hours,such as more than 12 hours, or even more than 24, 48 or 72 hours,compared to the corresponding amino acid sequence of the invention perse.

In a preferred, but non-limiting aspect of the invention, such compoundsor polypeptides of the invention have a serum half-life e.g. in humansthat is increased with more than 1 hours, preferably more than 2 hours,more preferably more than 6 hours, such as more than 12 hours, or evenmore than 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, suchcompounds or polypeptides of the invention exhibit a serum half-life inhuman of at least about 12 hours, preferably at least 24 hours, morepreferably at least 48 hours, even more preferably at least 72 hours ormore. For example, compounds or polypeptides of the invention may have ahalf-life of at least 5 days (such as about 5 to 10 days), preferably atleast 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), o/at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days).

In a particular preferred but non-limiting aspect of the invention, theinvention provides a polypeptide of the invention comprising i) one HGFbinding immunoglobulin single variable domain as described herein; andii) one or more (preferably one) serum albumin binding immunoglobulinsingle variable domain as described herein.

In a further preferred aspect, the invention provides a polypeptide ofthe invention comprising i) one HGF binding immunoglobulin singlevariable domain as described herein; and ii) one or more (preferablyone) serum albumin binding immunoglobulin single variable domain of SEQID NO: 114 or 115 (Table B-1).

In a further preferred aspect, the invention provides a polypeptide ofthe invention comprising i) one HGF binding immunoglobulin singlevariable domain as described herein; and ii) one or more (preferablyone) serum albumin binding immunoglobulin single variable domain withCDRs (defined according to the Kabat numbering) of SEQ ID NO: 114 or 115(Table B-1). Preferably, the invention relates to a serum albumin (SA)binding immunoglobulin single variable domain, which consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), wherein said CDR1 isSFGMS (SEQ ID NO: 128), said CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 129),and said CDR3 is GGSLSR (SEQ ID NO: 130).

Thus, for example, further reference (and thus incorporated byreference) is made in particular to the experimental part and furtherdescription of WO2008/058280, wherein further details on SEQ ID NO: 114or 115 is made and e.g., the half-life of a immunoglobulin singlevariable domain construct containing said sequence in rhesus monkeys isdisclosed.

These may comprise of two immunoglobulin single variable domains, suchas one immunoglobulin single variable domain directed against HGF andone immunoglobulin single variable domain against serum albumin. Suchmultispecific constructs will be clear to the skilled person based onthe disclosure herein; some preferred, but non-limiting examples of suchmultispecific immunoglobulin single variable domains are the constructsof SEQ ID NOs: 112 or 113 (see experimental part).

According to another specific, but non-limiting aspect, a polypeptide ofthe invention comprises or essentially consists of at least oneimmunoglobulin single variable domain of the invention and at least oneother binding unit (i.e., directed against another epitope, antigen,target, protein or polypeptide), which is preferably also animmunoglobulin single variable domain. Such proteins or polypeptides arealso referred to herein as “multispecific” proteins or polypeptides oras “multispecific constructs”, and these may comprise or consistessentially of two immunoglobulin single variable domains, such as oneimmunoglobulin single variable domain of the invention directed againstHGF and one immunoglobulin single variable domain against serum albumin.Such multispecific constructs will be clear to the skilled person basedon the disclosure herein; some preferred, but non-limiting examples ofsuch multispecific immunoglobulin single variable domains are theconstructs of SEQ ID NOs: 112 or 113 (see experimental part).

According to yet another specific, but non-limiting aspect, apolypeptide of the invention comprises or essentially consists of atleast one immunoglobulin single variable domain of the invention,optionally one or more further immunoglobulin single variable domains,and at least one other amino acid sequence (such as a protein orpolypeptide) that confers at least one desired property to theimmunoglobulin single variable domain of the invention and/or to theresulting fusion protein. Again, such fusion proteins may providecertain advantages compared to the corresponding monovalentimmunoglobulin single variable domains of the invention such as e.g.,may provide an increased half-life.

In the above constructs, the one or more immunoglobulin single variabledomains and/or other immunoglobulin single variable domains may bedirectly linked to each other and/or suitably linked to each other viaone or more linker sequences. Some suitable but non-limiting examples ofsuch linkers will become clear from the further description herein.

In one embodiment, the linker sequence joining the immunoglobulin singlevariable domains are described in Table B-5, e.g. SEQ ID NOs: 117 to126, or as known in the art.

In another preferred embodiment, the invention relates to a trispecific,or multispecific polypeptide, comprising or essentially consisting of atleast three ISVDs, wherein two of said at least three ISVDs are directedagainst a tumor associated antigen (such as, for instance, HGF and EGFRor VEGF) and the other binding moiety is directed against another targetor antigen. Preferably this target or antigen is a molecule which canincrease the half-life of the polypeptide in vivo (as further described)or a molecule with an effector function such as CD3, the Fc receptor ora complement protein.

In an embodiment, the invention provides trispecific polypeptidescomprising or essentially consisting of a Nanobody against EGFR or aNanobody against VEGF, a Nanobody against HGF and a Nanobody againsthuman serum albumin.

In another preferred embodiment, the invention relates to atetraspecific, or multispecific polypeptide, comprising or essentiallyconsisting of at least four ISVDs, wherein three of said at least fourISVDs are directed against a tumor associated antigen (such as, forinstance, HGF, EGFR and VEGF) and the other binding moiety is directedagainst another target or antigen. Preferably this target or antigen isa molecule which can increase the half-life of the polypeptide in vivo(as further described) or a molecule with an effector function such asCD3, the Fc receptor or a complement protein.

In an embodiment, the invention provides tetraspecific polypeptidescomprising or essentially consisting of a Nanobody against EGFR, aNanobody against VEGF, a Nanobody against HGF and a Nanobody againsthuman serum albumin.

Furthermore, although it is encompassed within the scope of theinvention that the specific order or arrangement of the variousNanobodies in the polypeptides of the invention may have some influenceon the properties of the final polypeptide of the invention (includingbut not limited to the affinity, specificity or avidity for VEGF, EGFRor HGF, respectively, or against the one or more other antigens), saidorder or arrangement is usually not critical and may be suitably chosenby the skilled person, optionally after some limited routine experimentsbased on the disclosure herein. Thus, when reference is made to aspecific multispecific polypeptide of the invention, it should be notedthat this encompasses any order or arrangements of the relevantNanobodies, unless explicitly indicated otherwise.

According to yet another specific, but non-limiting aspect, apolypeptide of the invention may for example be chosen from the groupconsisting of immunoglobulin single variable domains that have more than80%, preferably more than 90%, more preferably more than 95%, such as99% or more “sequence identity” (as defined herein) with one or more ofthe immunoglobulin single variable domains of SEQ ID NOs: 112 or 113(see experimental part), in which the polypeptides are preferably asfurther defined herein, i.e., in the preferred format of oneimmunoglobulin single variable domain directed against HGF and oneimmunoglobulin single variable domain directed against serum albumin.

According to yet another specific, but non-limiting aspect, apolypeptide of the invention may for example be chosen from the groupconsisting of polypeptides that have more than 80%, preferably more than90%, more preferably more than 95%, such as 99% or more “sequenceidentity” (as defined herein) with one or more of the polypeptides ofSEQ ID NOs: 6 to 27, preferably SEQ ID NOs: 7, 8, 10, 15, 16, 18 and20-25, even more preferably SEQ ID NOs: 7 and 18.

Generally, for pharmaceutical use, the polypeptides of the invention maybe formulated as a pharmaceutical preparation or composition comprisingat least one polypeptide of the invention and at least onepharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activepolypeptides and/or compounds. By means of non-limiting examples, such aformulation may be in a form suitable for oral administration, forparenteral administration (such as by intravenous, intramuscular orsubcutaneous injection or intravenous infusion), for topicaladministration, for administration by inhalation, by a skin patch, by animplant, by a suppository, etc., wherein the parenteral administrationis preferred. Such suitable administration forms—which may be solid,semi-solid or liquid, depending on the manner of administration—as wellas Methods and carriers for use in the preparation thereof, will beclear to the skilled person, and are further described herein. Such apharmaceutical preparation or composition will generally be referred toherein as a “pharmaceutical composition”. A pharmaceutical preparationor composition for use in a non-human organism will generally bereferred to herein as a “veterinary composition”.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one amino acid of the invention, atleast one polypeptide of the invention or at least one polypeptide ofthe invention and at least one suitable carrier, diluent or excipient(i.e., suitable for pharmaceutical use), and optionally one or morefurther active substances.

Generally, the polypeptides of the invention can be formulated andadministered in any suitable manner known per se. Reference is forexample made to the general background art cited above (and inparticular to WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867 andWO 08/020,079) as well as to the standard handbooks, such as Remington'sPharmaceutical Sciences, 18^(th) Ed., Mack Publishing Company, USA(1990), Remington, the Science and Practice of Pharmacy, 21st Edition,Lippincott Williams and Wilkins (2005); or the Handbook of TherapeuticAntibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see for example pages252-255).

The polypeptides of the invention may be formulated and administered inany manner known per se for conventional antibodies and antibodyfragments (including ScFv's and diabodies) and other pharmaceuticallyactive proteins. Such formulations and methods for preparing the samewill be clear to the skilled person, and for example includepreparations suitable for parenteral administration (e.g. intravenous,intraperitoneal, subcutaneous, intramuscular, intraluminal,intra-arterial or intrathecal administration) or for topical (i.e.,transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, those mentioned onpage 143 of WO 08/020,079. In one embodiment, the preparation is anaqueous solution or suspension.

The polypeptides of the invention can be administered using methods ofdelivery known from gene therapy, see, e.g., U.S. Pat. No. 5,399,346,which is incorporated by reference for its gene therapy deliverymethods. Using a gene therapy Method of delivery, primary cellstransfected with the gene encoding an amino acid sequence, polypeptideof the invention can additionally be transfected with tissue specificpromoters to target specific organs, tissue, grafts, tumors, or cellsand can additionally be transfected with signal and stabilizationsequences for subcellularly localized expression.

Thus, the polypeptides of the invention may be systemicallyadministered, e.g., orally, in combination with a pharmaceuticallyacceptable vehicle such as an inert diluent or an assimilable ediblecarrier. They may be enclosed in hard or soft shell gelatin capsules,may be compressed into tablets, or may be incorporated directly with thefood of the patient's diet. For oral therapeutic administration, thepolypeptides of the invention may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% of thepolypeptide of the invention. Their percentage in the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 60% of the weight of a given unit dosage form. Theamount of the polypeptide of the invention in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

For local administration at the site of tumor resection, thepolypeptides of the invention may be used in biodegradable polymericdrug delivery systems, slow release poly(lactic-co-glycolic acid)formulations and the like (Hart et al., Cochrane Database Syst Rev. 2008Jul. 16; (3): CD007294).

In a further preferred aspect of the invention, the polypeptides of theinvention, such as a polypeptide consisting essentially of onemonovalent anti-human HGF immunoglobulin single variable domain and ofone monovalent anti-human serum albumin immunoglobulin single variabledomain linked by a GS linker, may have a beneficial distribution andkinetics profile in solid tumors compared to conventional antibodies,such as, e.g. IgG.

The tablets, troches, pills, capsules, and the like may also containbinders, excipients, disintegrating agents, lubricants and sweetening orflavoring agents, for example those mentioned on pages 143-144 of WO08/020,079. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the polypeptides of the invention, sucrose orfructose as a sweetening agent, methyl- and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any unit dosage form should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed. In addition, the polypeptides of the invention may beincorporated into sustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The polypeptides of the invention may also be administered intravenouslyor intraperitoneally by infusion or injection. Particular examples areas further described on pages 144 and 145 of WO 08/020,079 or inPCT/EP2010/062975 (entire document).

For topical administration, the polypeptides of the invention may beapplied in pure form, i.e., when they are liquids. However, it willgenerally be desirable to administer them to the skin as compositions orformulations, in combination with a dermatologic acceptable carrier,which may be a solid or a liquid. Particular examples are as furtherdescribed on page 145 of WO 08/020,079.

Generally, the concentration of the polypeptides of the invention in aliquid composition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

The amount of the polypeptides of the invention required for use intreatment will vary not only with the particular polypeptide selectedbut also with the route of administration, the nature of the conditionbeing treated and the age and condition of the patient and will beultimately at the discretion of the attendant physician or clinician.Also the dosage of the polypeptides of the invention varies depending onthe target cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder associated withHGF, said method comprising administering, to a subject in need thereof,a pharmaceutically active amount of a polypeptide of the invention,and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease or disorder that is associated with HGF, with itsbiological or pharmacological activity, and/or with the biologicalpathways or signaling in which HGF is involved, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of an amino acid sequence of the invention, of a polypeptide ofthe invention and/or of a pharmaceutical composition comprising thesame. In an embodiment, the invention relates to a method for theprevention and/or treatment of at least one disease or disorder that canbe treated by modulating HGF, its biological or pharmacologicalactivity, and/or the biological pathways or signaling in which HGF isinvolved, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.In an embodiment, said pharmaceutically effective amount may be anamount that is sufficient to modulate HGF, its biological orpharmacological activity, and/or the biological pathways or signaling inwhich HGF is involved; and/or an amount that provides a level of thepolypeptide of the invention in the circulation that is sufficient tomodulate HGF, its biological or pharmacological activity, and/or thebiological pathways or signaling in which HGF is involved.

In an embodiment the invention relates to a method for the preventionand/or treatment of at least one disease or disorder that can beprevented and/or treated by administering a polypeptide of theinvention, or a nucleotide construct of the invention encoding the same,and/or of a pharmaceutical composition comprising the same, to apatient. In an embodiment, the method comprises administering apharmaceutically active amount of a polypeptide of the invention, or anucleotide construct of the invention encoding the same, and/or of apharmaceutical composition comprising the same to a subject in needthereof.

In an embodiment the invention relates to a method for the preventionand/or treatment of at least one disease or disorder that can beprevented and/or treated by inhibiting binding of HGF to c-Met inspecific cells or in a specific tissue of a subject to be treated (andin particular, by inhibiting binding of HGF to c-Met in cancer cells orin a tumor present in the subject to be treated), said method comprisingadministering a pharmaceutically active amount of a polypeptide of theinvention, or a nucleotide construct of the invention encoding the same,and/or of a pharmaceutical composition comprising the same, to a subjectin need thereof.

In an embodiment, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apolypeptide of the invention, or a nucleotide construct of the inventionencoding the same, and/or of a pharmaceutical composition comprising thesame.

In an embodiment, the invention relates to a method for immunotherapy,and in particular for passive immunotherapy, which method comprisesadministering, to a subject suffering from or at risk of the diseasesand disorders mentioned herein, a pharmaceutically active amount of apolypeptide of the invention, or a nucleotide construct of the inventionencoding the same, and/or of a pharmaceutical composition comprising thesame.

In the above methods, the amino acid sequences, polypeptides of theinvention and/or the compositions comprising the same can beadministered in any suitable manner, depending on the specificpharmaceutical formulation or composition to be used. Thus, thepolypeptides of the invention and/or the compositions comprising thesame can for example be administered orally, intraperitoneally (e.g.intravenously, subcutaneously, intramuscularly, or via any other routeof administration that circumvents the gastrointestinal tract),intranasally, transdermally, topically, by means of a suppository, byinhalation, again depending on the specific pharmaceutical formulationor composition to be used. The clinician will be able to select asuitable route of administration and a suitable pharmaceuticalformulation or composition to be used in such administration, dependingon the disease or disorder to be prevented or treated and other factorswell known to the clinician.

The polypeptides of the invention and/or the compositions comprising thesame are administered according to a regime of treatment that issuitable for preventing and/or treating the disease or disorder to beprevented or treated. The clinician will generally be able to determinea suitable treatment regimen, depending on factors such as the diseaseor disorder to be prevented or treated, the severity of the disease tobe treated and/or the severity of the symptoms thereof, the polypeptideof the invention to be used, the specific route of administration andpharmaceutical formulation or composition to be used, the age, gender,weight, diet, general condition of the patient, and similar factors wellknown to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more polypeptides of the invention, or of one or more compositionscomprising the same, in one or more pharmaceutically effective amountsor doses. The specific amount(s) or doses to be administered can bedetermined by the clinician, again based on the factors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific polypeptide of theinvention to be used, the specific route of administration and thespecific pharmaceutical formulation or composition used, thepolypeptides of the invention will generally be administered in anamount between 1 gram and 0.01 microgram per kg body weight per day,preferably between 0.1 gram and 0.1 microgram per kg body weight perday, such as about 1, 10, 100 or 1000 microgram per kg body weight perday, either continuously (e.g. by infusion), as a single daily dose oras multiple divided doses during the day. The clinician will generallybe able to determine a suitable daily dose, depending on the factorsmentioned herein. It will also be clear that in specific cases, theclinician may choose to deviate from these amounts, for example on thebasis of the factors cited above and his expert judgment. Generally,some guidance on the amounts to be administered can be obtained from theamounts usually administered for comparable conventional antibodies orantibody fragments against the same target administered via essentiallythe same route, taking into account however differences inaffinity/avidity, efficacy, biodistribution, half-life and similarfactors well known to the skilled person.

In an embodiment, a single contiguous polypeptide of the invention willbe used. In one embodiment two or more polypeptides of the invention areprovided in combination.

The polypeptides of the invention may be used in combination with one ormore further pharmaceutically active compounds or principles, i.e., as acombined treatment regimen, which may or may not lead to a synergisticeffect. Again, the clinician will be able to select such furthercompounds or principles, as well as a suitable combined treatmentregimen, based on the factors cited above and his expert judgment.

In particular, the polypeptides of the invention may be used incombination with other pharmaceutically active compounds or principlesthat are or can be used for the prevention and/or treatment of thediseases and disorders cited herein, as a result of which a synergisticeffect may or may not be obtained. Examples of such compounds andprinciples, as well as routes, methods and pharmaceutical formulationsor compositions for administering them will be clear to the clinician,and generally include the cytostatic and preferably cytotoxic activeprinciples usually applied for the treatment of the tumor to be treated.

Specifically contemplated combinations for use with the polypeptides ofthe invention for oncology include, but are not limited to, e.g., RONantagonists, CXCR4 antagonists such as e.g. AMD3100, other chemokinereceptor antagonists, taxol; gemcitabine; cisplatin; cIAP inhibitors(such as inhibitors to cIAP1, cIAP2 and/or XIAP); MEK inhibitorsincluding but not limited to, e.g., U0126, PD0325901; bRaf inhibitorsincluding but not limited to, e.g., RAF265; and mTOR inhibitorsincluding but not limited to, e.g., RAD001; VEGF inhibitors includingbut not limited to e.g. bevacizumab, sutinib and sorafenib; ERBBinhibitors, such as, for instance, EGFR-inhibitors, including but notlimited to specific small molecule kinase inhibitors, e.g. erlotinib,gefitinib; antibodies, e.g. cetuximab, nimotuzumab, panitumumab,necitumumab, IMC-C225 (Erbitux, Imclone), EMD72000 (Merck Darmstadt),ABX-EGF (Abgenix), h-R3 (theraCIM, YM Biosciences) and Humax-EGFR(Genmab); dual- or multispecific small molecule kinase inhibitors, e.g.lapatinib (EGFR&HER2), vandetanib (EGFR, RET, VEGFR2), neratinib (EGFR,HER2, HER4) and PF-299804 (EGFR, HER2, HER4), HER2-inhibitors includingbut not limited to e.g. trastuzumab and lapatinib; HER3-inhibitors; HER4inhibitors; PDGFR, FGFR, src, JAK, STAT and/or GSK3 inhibitors;selective estrogen receptor modulators including but not limited totamoxifen; estrogen receptor downregulators including but not limited tofulvestrant. Specific contemplated combinations for use with thepolypeptides of the invention for e.g. inflammatory and other conditionsalso include, but are not limited to, e.g., interferon beta 1 alpha andbeta, IFN alpha 2b; natalizumab; TNF alpha antagonists including but notlimited to e.g. infliximab, adalimumab, certolizumab pegol, etanercept;disease-modifying antirheumatic drugs such as e.g. methotrexate (MTX);glucocorticoids including but not limited to e.g. dexamethasone,hydrocortisone; nonsteroidal anti-inflammatory drugs including but notlimited to e.g. ibuprofen, sulindac; IL-6 or IL-6R inhibitors includingbut not limited to e.g. RoActemra, ALD518. In addition combinations foruse with the polypeptides of the invention for oncology indicationsinclude but are not limited to non-targeted chemotherapeutics such ascytotoxics and/or cytostatics. The invention also comprises productsand/or compositions comprising the polypeptides of the invention incombination with other antibodies and/or chemical compounds directedagainst other growth factors involved in tumor progression or metastasisand/or compounds and/or anticancer agents or agents conjugated withtoxins and their use for the prevention and/or the treatment of certaincancers.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of polypeptide ofthe invention in the preparation of a pharmaceutical composition forprevention and/or treatment of at least one disease and disorderassociated with HGF; and/or for use in one or more of the methods oftreatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. In veterinaryapplications, the subject to be treated includes any animal raised forcommercial purposes or kept as a pet. As will be clear to the skilledperson, the subject to be treated will in particular be a personsuffering from, or at risk of, the diseases and disorders mentionedherein.

The invention relates to the use of a polypeptide of the invention, or anucleotide encoding the same, in the preparation of a pharmaceuticalcomposition for the prevention and/or treatment of at least one diseaseor disorder that can be prevented and/or treated by administering apolypeptide of the invention, or a nucleotide encoding the same, and/ora pharmaceutical composition of the same to a patient.

More in particular, the invention relates to the use of a polypeptide ofthe invention, or a nucleotide encoding the same, in the preparation ofa pharmaceutical composition for the prevention and/or treatment ofdiseases and disorders associated with HGF, and in particular for theprevention and treatment of one or more of the diseases and disorderslisted herein.

Again, in such a pharmaceutical composition, the one or morepolypeptide(s) of the invention, or nucleotide(s) encoding the same,and/or a pharmaceutical composition of the same, may also be suitablycombined with one or more other active principles, such as thosementioned herein.

The invention also relates to a composition (such as, withoutlimitation, a pharmaceutical composition or preparation as furtherdescribed herein) for use, either in vitro (e.g. in an in vitro orcellular assay) or in vivo (e.g. in an a single cell or multi-cellularorganism, and in particular in a mammal, and more in particular in ahuman being, such as in a human being that is at risk of or suffers froma disease or disorder of the invention).

In the context of the present invention, “modulating” or “to modulate”generally means reducing or inhibiting the activity of HGF and inparticular human HGF (SEQ ID NO: 1), as measured using a suitable invitro, cellular or in vivo assay (such as those mentioned herein). Inparticular, reducing or inhibiting the activity of HGF and in particularhuman HGF (SEQ ID NO: 1), as measured using a suitable in vitro,cellular or in vivo assay (such as those mentioned herein), by at least1%, preferably at least 5%, such as at least 10% or at least 25%, forexample by at least 50%, at least 60%, at least 70%, at least 80%, or90% or more, compared to activity of HGF and in particular human HGF(SEQ ID NO: 1) in the same assay under the same conditions but withoutthe presence of the polypeptide of the invention.

Modulating may for example involve reducing or inhibiting the binding ofHGF to one of its substrates or receptors and/or competing with otherligands, substrate for binding to c-Met. Alternatively, modulating mayinvolve inhibiting the internalization, inducing internalization inorder to reduce c-Met level and as such reducing signaling,homodimerization of c-Met and/or promoting of shedding of c-Met and thusmay inhibit HGF dependent c-Met activation.

The invention further relates to methods for preparing or generating theimmunoglobulin single variable domains, polypeptides, nucleic acids,host cells, products and compositions described herein. Some preferredbut non-limiting examples of such methods will become clear from thefurther description herein.

Generally, these methods may comprise the steps of:

-   a) providing a set, collection or library of immunoglobulin single    variable domains; and-   b) screening said set, collection or library of immunoglobulin    single variable domains for immunoglobulin single variable domains    that can bind to and/or have affinity for HGF and in particular    human HGF (SEQ ID NO: 1); and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for HGF and in particular human HGF (SEQ ID NO: 1).

In such a method, the set, collection or library of immunoglobulinsingle variable domains may be any suitable set, collection or libraryof immunoglobulin single variable domains. For example, the set,collection or library of immunoglobulin single variable domains may be aset, collection or library of immunoglobulin sequences (as describedherein), such as a naive set, collection or library of immunoglobulinsequences; a synthetic or semi-synthetic set, collection or library ofimmunoglobulin sequences; and/or a set, collection or library ofimmunoglobulin sequences that have been subjected to affinitymaturation.

Also, in such a method, the set, collection or library of immunoglobulinsingle variable domains may be a set, collection or library of heavy orlight chain variable domains (such as VL-, VH- or VHH domains,preferably VHH domains). For example, the set, collection or library ofimmunoglobulin single variable domains may be a set, collection orlibrary of domain antibodies or single domain antibodies, or may be aset, collection or library of immunoglobulin single variable domainsthat are capable of functioning as a domain antibody or single domainantibody.

In a preferred aspect of this method, the set, collection or library ofimmunoglobulin single variable domains may be an immune set, collectionor library of immunoglobulin sequences, for example derived from amammal that has been suitably immunized with HGF and in particular humanHGF (SEQ ID NO: 1) or with a suitable antigenic determinant basedthereon or derived there from, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

In the above methods, the set, collection or library of immunoglobulinsingle variable domains may be displayed on a phage, phagemid, ribosomeor suitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of)immunoglobulin single variable domains will be clear to the personskilled in the art, for example on the basis of the further disclosureherein. Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23:1105-1116 (2005).

In another aspect, the method for generating immunoglobulin singlevariable domains comprises at least the steps of:

-   a) providing a collection or sample of cells expressing    immunoglobulin single variable domains;-   b) screening said collection or sample of cells for cells that    express an amino acid sequence that can bind to and/or have affinity    for HGF and in particular human HGF (SEQ ID NO: 1); and-   c) either (i) isolating said amino acid sequence; or (ii) isolating    from said cell a nucleic acid sequence that encodes said amino acid    sequence, followed by expressing said amino acid sequence.

In another aspect, the method for generating an amino acid sequencedirected against HGF and in particular human HGF (SEQ ID NO: 1) maycomprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding immunoglobulin single variable domains;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for HGF and in    particular human HGF (SEQ ID NO: 1); and    -   c) isolating said nucleic acid sequence, followed by expressing        said amino acid sequence.

In such a method, the set, collection or library of nucleic acidsequences encoding immunoglobulin single variable domains may forexample be a set, collection or library of nucleic acid sequencesencoding a naive set, collection or library of immunoglobulin sequences;a set, collection or library of nucleic acid sequences encoding asynthetic or semi-synthetic set, collection or library of immunoglobulinsequences; and/or a set, collection or library of nucleic acid sequencesencoding a set, collection or library of immunoglobulin sequences thathave been subjected to affinity maturation.

In another aspect, the method for generating an amino acid sequencedirected against HGF and in particular human HGF (SEQ ID NO: 1) maycomprise at least the steps of:

-   -   a) providing a set, collection or library of nucleic acid        sequences encoding immunoglobulin single variable domains;    -   b) screening said set, collection or library of nucleic acid        sequences for nucleic acid sequences that encode an amino acid        sequence that can bind to and/or has affinity for HGF and in        particular human HGF (SEQ ID NO: 1) and that is cross-blocked or        is cross blocking a immunoglobulin single variable domain or        polypeptide of the invention, e.g. SEQ ID NOs: 6 to 27,        preferably SEQ ID NOs: 7, 8, 10, 15, 16, 18 and 20-25, even more        preferably SEQ ID NOs: 7 and 18; and

-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

In preferred aspect, the method for generating an amino acid sequencedirected against HGF and in particular human HGF (SEQ ID NO: 1) maycomprise at least the steps of:

-   a) providing a set, collection or library of immunoglobulin single    variable domains; and-   b) screening said set, collection or library of immunoglobulin    single variable domains for immunoglobulin single variable domains    that can bind to and/or have affinity for HGF and in particular    human HGF (SEQ ID NO: 1); and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for HGF and in particular human HGF (SEQ ID NO: 1).

In such a method, the set, collection or library of immunoglobulinsingle variable domains may be any suitable set, collection or libraryof immunoglobulin single variable domains. For example, the set,collection or library of immunoglobulin single variable domains may be aset, collection or library of immunoglobulin sequences (as describedherein), such as a naive set, collection or library of immunoglobulinsequences; a synthetic or semi-synthetic set, collection or library ofimmunoglobulin sequences; and/or a set, collection or library ofimmunoglobulin sequences that have been subjected to affinitymaturation. In a preferred aspect, the set, collection or library ofimmunoglobulin single variable domains may be a set, collection orlibrary of immunoglobulin sequences (as described herein), such as asynthetic set, collection or library of immunoglobulin sequences. In theabove methods, the set, collection or library of immunoglobulin singlevariable domains may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of)immunoglobulin single variable domains will be clear to the personskilled in the art such as e.g. described by Knappik et al., J. Mol.Biol. 2000 Feb. 11, 296:57-86.

Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) immunoglobulin singlevariable domains will be clear to the person skilled in the art, forexample on the basis of the further disclosure herein. Reference is alsomade to the review by Hoogenboom in Nature Biotechnology, 23:1105-1116(2005).

The invention also relates to immunoglobulin single variable domainsthat are obtained by the above methods, or alternatively by a methodthat comprises the one of the above methods and in addition at least thesteps of determining the nucleotide sequence or amino acid sequence ofsaid immunoglobulin sequence; and of expressing or synthesizing saidamino acid sequence in a manner known per se, such as by expression in asuitable host cell or host organism or by chemical synthesis.

Also, following the steps above, one or more immunoglobulin singlevariable domains of the invention may be suitably humanized, camelizedor otherwise sequence optimized (e.g. sequence optimized formanufacturability, stability and/or solubility); and/or the amino acidsequence(s) thus obtained may be linked to each other or to one or moreother suitable immunoglobulin single variable domains (optionally viaone or more suitable linkers) so as to provide a polypeptide of theinvention. Also, a nucleic acid sequence encoding an amino acid sequenceof the invention may be suitably humanized, camelized or otherwisesequence optimized (e.g. sequence optimized for manufacturability,stability and/or solubility) and suitably expressed; and/or one or morenucleic acid sequences encoding an amino acid sequence of the inventionmay be linked to each other or to one or more nucleic acid sequencesthat encode other suitable immunoglobulin single variable domains(optionally via nucleotide sequences that encode one or more suitablelinkers), after which the nucleotide sequence thus obtained may besuitably expressed so as to provide a polypeptide of the invention.

The invention further relates to applications and uses of theimmunoglobulin single variable domains, compounds, constructs,polypeptides, nucleic acids, host cells, products and compositionsdescribed herein, as well as to methods for the diagnosis, preventionand/or treatment for diseases and disorders associated with HGF and inparticular human HGF (SEQ ID NO: 1). Some preferred but non-limitingapplications and uses will become clear from the further descriptionherein.

The invention also relates to the immunoglobulin single variabledomains, compounds, constructs, polypeptides, nucleic acids, host cells,products and compositions described herein for use in therapy.

In particular, the invention also relates to the immunoglobulin singlevariable domains, compounds, constructs, polypeptides, nucleic acids,host cells, products and compositions described herein for use intherapy of a disease or disorder that can be prevented or treated byadministering, to a subject in need thereof, of (a pharmaceuticallyeffective amount of) an amino acid sequence, compound, construct, ISVDor polypeptide as described herein.

More in particular, the invention relates to the immunoglobulin singlevariable domains, compounds, constructs, polypeptides, nucleic acids,host cells, products and compositions described herein for use intherapy of cancer.

Polypeptides of the invention and immunoglobulin single variable domains(that form part of the polypeptides of the invention) may be altered inorder to further improve potency or other desired properties.

Generally, an immunoglobulin single variable domain can be defined as apolypeptide with the formula 1

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively (cf. above).

Some particularly preferred, but non-limiting combinations of CDRsequences, as well as preferred combinations of CDR sequences andframework sequences, are mentioned in Table B-2 below, which lists theCDR sequences and framework sequences that are present in a number ofpreferred (but non-limiting) immunoglobulin single variable domains ofthe invention. As will be clear to the skilled person, a combination ofCDR1, CDR2 and CDR3 sequences that occur in the same clone (i.e., CDR1,CDR2 and CDR3 sequences that are mentioned on the same line or row inTable B-2) will usually be preferred (although the invention in itsbroadest sense is not limited thereto, and also comprises other suitablecombinations of the CDR sequences mentioned in Table B-2). Also, acombination of CDR sequences and framework sequences that occur in thesame clone (i.e. CDR sequences and framework sequences that arementioned on the same line or row in Table B-2) will usually bepreferred (although the invention in its broadest sense is not limitedthereto, and also comprises other suitable combinations of the CDRsequences and framework sequences mentioned in Table B-2, as well ascombinations of such CDR sequences and other suitable frameworksequences, e.g., as further described herein).

Also, in the immunoglobulin single variable domains of the inventionthat comprise the combinations of CDRs mentioned in Table B-2, each CDRcan be replaced by a CDR chosen from the group consisting ofimmunoglobulin single variable domains that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity (as defined herein) with thementioned CDRs, in which:

-   i) any amino acid substitution in such a CDR is preferably, and    compared to the corresponding CDR sequence mentioned in Table B-2, a    conservative amino acid substitution (as defined herein); and/or-   ii) any such CDR sequence preferably only contains amino acid    substitutions, and no amino acid deletions or insertions, compared    to the corresponding CDR sequence mentioned in Table B-2; and/or-   iii) any such CDR sequence is a CDR that is derived by means of a    technique for affinity maturation known per se, and in particular    starting from the corresponding CDR sequence mentioned in Table B-2.

However, as will be clear to the skilled person, the (combinations of)CDR sequences, as well as (the combinations of) CDR sequences andframework sequences mentioned in Table B-2 will generally be preferred.

Thus, in the immunoglobulin single variable domains of the invention, atleast one of the CDR1, CDR2 and CDR3 sequences present is suitablychosen from the group consisting of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-2; or from the group of CDR1, CDR2 andCDR3 sequences, respectively, that have at least 80%, preferably atleast 90%, more preferably at least 95%, even more preferably at least99% “sequence identity” (as defined herein) with at least one of theCDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-2; and/orfrom the group consisting of the CDR1, CDR2 and CDR3 sequences,respectively, that have 3, 2 or only 1 “amino acid difference(s)” (asdefined herein) with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-2.

In this context, by “suitably chosen” is meant that, as applicable, aCDR1 sequence is chosen from suitable CDR1 sequences (i.e., as definedherein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e.,as defined herein), and a CDR3 sequence is chosen from suitable CDR3sequence (i.e. as defined herein), respectively. More in particular, theCDR sequences are preferably chosen such that the immunoglobulin singlevariable domains of the invention bind to HGF and in particular humanHGF (SEQ ID NO: 1) with an affinity (suitably measured and/or expressedas a EC50 value, or alternatively as an IC₅₀ value, as further describedherein in various in vitro and/or in vivo potency or other assays) thatis as defined herein.

In particular, in the immunoglobulin single variable domains of theinvention, at least the CDR3 sequence present is suitably chosen fromthe group consisting of the CDR3 sequences listed in Table B-2 or fromthe group of CDR3 sequences that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with at least one of the CDR3 sequences listed inTable B-2; and/or from the group consisting of the CDR3 sequences thathave 3, 2 or only 1 amino acid difference(s) with at least one of theCDR3 sequences listed in Table B-2.

Preferably, in the immunoglobulin single variable domains of theinvention, at least two of the CDR1, CDR2 and CDR3 sequences present aresuitably chosen from the group consisting of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-2 or from the groupconsisting of CDR1, CDR2 and CDR3 sequences, respectively, that have atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 99% sequence identity with at least one of theCDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-2; and/orfrom the group consisting of the CDR1, CDR2 and CDR3 sequences,respectively, that have 3, 2 or only 1 “amino acid difference(s)” withat least one of the CDR1, CDR2 and CDR3 sequences, respectively, listedin Table B-2.

In particular, in the immunoglobulin single variable domains of theinvention, at least the CDR3 sequence present is suitably chosen fromthe group consisting of the CDR3 sequences listed in Table B-2 or fromthe group of CDR3 sequences that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with at least one of the CDR3 sequences listed inTable B-2, respectively; and at least one of the CDR1 and CDR2 sequencespresent is suitably chosen from the group consisting of the CDR1 andCDR2 sequences, respectively, listed in Table B-2 or from the group ofCDR1 and CDR2 sequences, respectively, that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with at least one of the CDR1and CDR2 sequences, respectively, listed in Table B-2; and/or from thegroup consisting of the CDR1 and CDR2 sequences, respectively, that have3, 2 or only 1 amino acid difference(s) with at least one of the CDR1and CDR2 sequences, respectively, listed in Table B-2.

Most preferably, in the immunoglobulin single variable domains of theinvention, all three CDR1, CDR2 and CDR3 sequences present are suitablychosen from the group consisting of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-2 or from the group of CDR1, CDR2 andCDR3 sequences, respectively, that have at least 80%, preferably atleast 90%, more preferably at least 95%, even more preferably at least99% sequence identity with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-2; and/or from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have3, 2 or only 1 amino acid difference(s) with at least one of the CDR1,CDR2 and CDR3 sequences, respectively, listed in Table B-2.

Even more preferably, in the immunoglobulin single variable domains ofthe invention, at least one of the CDR1, CDR2 and CDR3 sequences presentis suitably chosen from the group consisting of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-2. Preferably, in thisaspect, at least one or preferably both of the other two CDR sequencespresent are suitably chosen from CDR sequences that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with at least one of thecorresponding CDR sequences, respectively, listed in Table B-2; and/orfrom the group consisting of the CDR sequences that have 3, 2 or only 1amino acid difference(s) with at least one of the correspondingsequences, respectively, listed in Table B-2.

In particular, in the immunoglobulin single variable domains of theinvention, at least the CDR3 sequence present is suitably chosen fromthe group consisting of the CDR3 listed in Table B-2. Preferably, inthis aspect, at least one and preferably both of the CDR1 and CDR2sequences present are suitably chosen from the groups of CDR1 and CDR2sequences, respectively, that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with the CDR1 and CDR2 sequences, respectively, listedin Table B-2; and/or from the group consisting of the CDR1 and CDR2sequences, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2 sequences,respectively, listed in Table B-2.

Even more preferably, in the immunoglobulin single variable domains ofthe invention, at least two of the CDR1, CDR2 and CDR3 sequences presentare suitably chosen from the group consisting of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-2. Preferably, in thisaspect, the remaining CDR sequence present is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the corresponding CDR sequences listed inTable B-2; and/or from the group consisting of CDR sequences that have3, 2 or only 1 amino acid difference(s) with at least one of thecorresponding sequences listed in Table B-2.

In particular, in the immunoglobulin single variable domains of theinvention, at least the CDR3 sequence is suitably chosen from the groupconsisting of the CDR3 sequences listed in Table B-2, and either theCDR1 sequence or the CDR2 sequence is suitably chosen from the groupconsisting of the CDR1 and CDR2 sequences, respectively, listed in TableB-2. Preferably, in this aspect, the remaining CDR sequence present issuitably chosen from the group of CDR sequences that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with at least one of thecorresponding CDR sequences listed in Table B-2; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with the corresponding CDR sequences listed in Table B-2.

Even more preferably, in the immunoglobulin single variable domains ofthe invention, all three CDR1, CDR2 and CDR3 sequences present aresuitably chosen from the group consisting of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-2.

Also, generally, the combinations of CDR's listed in Table B-2 (i.e.,those mentioned on the same line or row in Table B-2) are preferred.Thus, it is generally preferred that, when a CDR in a immunoglobulinsingle variable domain of the invention is a CDR sequence mentioned inTable B-2 or is suitably chosen from the group of CDR sequences thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with a CDRsequence listed in Table B-2; and/or from the group consisting of CDRsequences that have 3, 2 or only 1 amino acid difference(s) with a CDRsequence listed in Table B-2, that at least one and preferably both ofthe other CDR's are suitably chosen from the CDR sequences that belongto the same combination in Table B-2 (i.e., mentioned on the same lineor now in Table B-2) or are suitably chosen from the group of CDRsequences that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with the CDR sequence(s) belonging to the same combinationand/or from the group consisting of CDR sequences that have 3, 2 or only1 amino acid difference(s) with the CDR sequence(s) belonging to thesame combination. The other preferences indicated in the aboveparagraphs also apply to the combinations of CDRs mentioned in TableB-2, e.g., mentioned on the same row in Table B-2.

Thus, by means of non-limiting examples, a polypeptide of the inventioncan for example comprise a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table B-2, a CDR2sequence that has 3, 2 or 1 amino acid difference with one of the CDR2sequences mentioned in Table B-2 (but belonging to a differentcombination, e.g., mentioned on different rows in Table B-2), and a CDR3sequence.

Some preferred immunoglobulin single variable domains of the inventionmay for example comprise: (1) a CDR1 sequence that has more than 80%sequence identity with one of the CDR1 sequences mentioned in Table B-2;a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of theCDR2 sequences mentioned in Table B-2 (but belonging to a differentcombination, e.g. mentioned on different rows in Table B-2); and a CDR3sequence that has more than 80% sequence identity with one of the CDR3sequences mentioned in Table B-2 (but belonging to a differentcombination, e.g. mentioned on different rows in Table B-2); or (2) aCDR1 sequence that has more than 80% sequence identity with one of theCDR1 sequences mentioned in Table B-2; a CDR2 sequence, and one of theCDR3 sequences listed in Table B-2; or (3) a CDR1 sequence; a CDR2sequence that has more than 80% sequence identity with one of the CDR2sequence listed in Table B-2; and a CDR3 sequence that has 3, 2 or 1amino acid differences with the CDR3 sequence mentioned in Table B-2that belongs to the same combination as the CDR2 sequence, e.g.,mentioned on the same rows in Table B-2.

Some particularly preferred immunoglobulin single variable domains ofthe invention may for example comprise: (1) a CDR1 sequence that hasmore than 80% sequence identity with one of the CDR1 sequences mentionedin Table B-2; a CDR2 sequence that has 3, 2 or 1 amino acid differencewith the CDR2 sequence mentioned in Table B-2 that belongs to the samecombination; and a CDR3 sequence that has more than 80% sequenceidentity with the CDR3 sequence mentioned in Table B-2 that belongs tothe same combination; (2) a CDR1 sequence; a CDR2 listed in Table B-2and a CDR3 sequence listed in Table B-2 (in which the CDR2 sequence andCDR3 sequence may belong to different combinations).

Some even more preferred immunoglobulin single variable domains of theinvention may for example comprise: (1) a CDR1 sequence that has morethan 80% sequence identity with one of the CDR1 sequences mentioned inTable B-2; the CDR2 sequence listed in Table B-1 that belongs to thesame combination; and a CDR3 sequence mentioned in Table B-2 thatbelongs to a different combination (e.g. mentioned on different rows inTable B-2); or (2) a CDR1 sequence mentioned in Table B-2; a CDR2sequence that has 3, 2 or 1 amino acid differences with the CDR2sequence mentioned in Table B-2 that belongs to the same combination;and a CDR3 sequence that has more than 80% sequence identity with theCDR3 sequence listed in Table B-2 that belongs to the same or adifferent combination.

Particularly preferred immunoglobulin single variable domains of theinvention may for example comprise a CDR1 sequence mentioned in TableB-2, a CDR2 sequence that has more than 80% sequence identity with theCDR2 sequence mentioned in Table B-2 that belongs to the samecombination; and the CDR3 sequence mentioned in Table B-2 that belongsto the same combination.

In an even more preferred immunoglobulin single variable domains of theinvention, the CDR1, CDR2 and CDR3 sequences present are suitably chosenfrom one of the combinations of CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-2. Most preferably CDR1 is SEQ ID NO:40.

According to another preferred, but non-limiting aspect of the invention(a) CDR1 has a length of between 1 and 12 amino acid residues, andusually between 2 and 9 amino acid residues, such as 5, 6 or 7 aminoacid residues; and/or (b) CDR2 has a length of between 13 and 24 aminoacid residues, and usually between 15 and 21 amino acid residues, suchas 16 and 17 amino acid residues; and/or (c) CDR3 has a length ofbetween 2 and 35 amino acid residues, and usually between 3 and 30 aminoacid residues, such as between 6 and 23 amino acid residues.

In another preferred, but non-limiting aspect, the invention relates toa immunoglobulin single variable domain in which the CDR sequences (asdefined herein) have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more sequence identity (asdefined herein) with the CDR sequences of at least one of theimmunoglobulin single variable domains of SEQ ID NOs: 6 to 27,preferably SEQ ID NOs: 7, 8, 10, 15, 16, 18 and 20-25, even morepreferably SEQ ID NOs: 7 and 18.

Another preferred, but non-limiting aspect of the invention relates tohumanized variants of the immunoglobulin single variable domains of SEQID NOs: 6 to 27, preferably SEQ ID NOs: 7, 8, 10, 15, 16, 18 and 20-25,even more preferably SEQ ID NOs: 7 and 18, that comprise, compared tothe corresponding native V_(HH) sequence, at least one humanizingsubstitution (as defined herein), and in particular at least onehumanizing substitution in at least one of its framework sequences (asdefined herein).

It will be clear to the skilled person that the immunoglobulin singlevariable domains that are mentioned herein as “preferred” (or “morepreferred”, “even more preferred”, etc.) are also preferred (or morepreferred, or even more preferred, etc.) for use in the polypeptidesdescribed herein. Thus, polypeptides that comprise or essentiallyconsist of one or more “preferred” immunoglobulin single variabledomains of the invention will generally be preferred, and polypeptidesthat comprise or essentially consist of one or more “more preferred”immunoglobulin single variable domains of the invention will generallybe more preferred, etc.

Another aspect of this invention relates to a nucleic acid that encodesan amino acid sequence of the invention (such as an immunoglobulinsingle variable domain of the invention) or a polypeptide of theinvention comprising the same. Again, as generally described herein forthe nucleic acids of the invention, such a nucleic acid may be in theform of a genetic construct, as defined herein.

In another preferred, but non-limiting aspect, the invention relates tonucleic acid sequences of immunoglobulin single variable domain in whichthe sequences (as defined herein) have more than 80%, preferably morethan 90%, more preferably more than 95%, such as 99% or more sequenceidentity (as defined herein) with the sequences of at least one ofnucleic acid sequence encoding the immunoglobulin single variabledomains SEQ ID NOs: 6 to 27, preferably SEQ ID NOs: 7, 8, 10, 15, 16, 18and 20-25, even more preferably SEQ ID NOs: 7 and 18.

In another aspect, the invention relates to nucleic acid sequences thatcomprise the nucleic acid sequences of immunoglobulin single variabledomain in which the sequences (as defined herein) have more than 80%,preferably more than 90%, more preferably more than 95%, such as 99% ormore sequence identity (as defined herein) with the sequences of atleast one of nucleic acid sequence encoding the immunoglobulin singlevariable domains SEQ ID NOs: 6 to 27, preferably SEQ ID NOs: 7, 8, 10,15, 16, 18 and 20-25, even more preferably SEQ ID NOs: 7 and 18.

In another aspect, the invention relates to a host or host cell whichexpresses or that is capable of expressing an amino acid sequence (suchas an immunoglobulin single variable domain) of the invention and/or apolypeptide of the invention comprising the same; and/or which containsa nucleic acid of the invention. Some preferred but non-limitingexamples of such hosts or host cells will become clear from the furtherdescription herein.

As will be clear to the skilled person, one particularly useful methodfor preparing a polypeptide of the invention generally comprises thesteps of:

-   i) the expression, in a suitable host cell or host organism (also    referred to herein as a “host of the invention”) or in another    suitable expression system of a nucleic acid that encodes said amino    acid sequence, polypeptide of the invention (also referred to herein    as a “nucleic acid of the invention”), optionally followed by:-   ii) isolating and/or purifying the polypeptide of the invention thus    obtained.

In particular, such a method may comprise the steps of:

-   i) cultivating and/or maintaining a host of the invention under    conditions that are such that said host of the invention expresses    and/or produces at least one polypeptide of the invention;    optionally followed by:-   ii) isolating and/or purifying the polypeptide of the invention thus    obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one aspect of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the immunoglobulinsingle variable domains for the polypeptides of the invention givenherein, and/or can be isolated from a suitable natural source. Toprovide analogs, nucleotide sequences encoding naturally occurringV_(HH) domains can for example be subjected to site-directedmutagenesis, so at to provide a nucleic acid of the invention encodingsaid analog. Also, as will be clear to the skilled person, to prepare anucleic acid of the invention, also several nucleotide sequences, suchas at least one nucleotide sequence encoding a polypeptide of theinvention and for example nucleic acids encoding one or more linkers canbe linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, hut are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g., to create cassettes and/or regions thatmay easily be digested and/or ligated using suitable restrictionenzymes), and/or the introduction of mutations by means of a PCRreaction using one or more “mismatched” primers, using for example asequence of a naturally occurring form of HGF and in particular humanHGF (SEQ ID NO: 1) as a template. These and other techniques will beclear to the skilled person, and reference is again made to the standardhandbooks, such as Sambrook et al. and Ausubel et al., mentioned above,as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art and as described on pages 131-134 of WO 08/020,079(incorporated herein by reference). Such genetic constructs generallycomprise at least one nucleic acid of the invention that is optionallylinked to one or more elements of genetic constructs known per se, suchas for example one or more suitable regulatory elements (such as asuitable promoter(s), enhancer(s), terminator(s), etc.) and the furtherelements of genetic constructs referred to herein. Such geneticconstructs comprising at least one nucleic acid of the invention willalso be referred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism. For instance, the genetic constructs of the invention maybe in the form of a vector, such as for example a plasmid, cosmid, YAC,a viral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g., in a suitable host cell, host organismand/or expression system).

In a preferred but non-limiting aspect, a genetic construct of theinvention comprises

-   i) at least one nucleic acid of the invention; operably connected to-   ii) one or more regulatory elements, such as a promoter and    optionally a suitable terminator; and optionally also-   iii) one or more further elements of genetic constructs known per    se;

in which the terms “operably connected” and “operably linked” have themeaning given on pages 131-134 of WO 08/020,079; and in which the“regulatory elements”, “promoter”, “terminator” and “further elements”are as described on pages 131-134 of WO 08/020,079; and in which thegenetic constructs may further be as described on pages 131-134 of WO08/020,079.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the polypeptide of the invention.Suitable hosts or host cells will be clear to the skilled person, andmay for example be any suitable fungal, prokaryotic or eukaryotic cellor cell line or any suitable fungal, prokaryotic or eukaryotic organism,for example those described on pages 134 and 135 of WO 08/020,079, aswell as all other hosts or host cells known per se for the expressionand production of antibodies and antibody fragments (including but notlimited to (single) domain antibodies and ScFv fragments), which will beclear to the skilled person. Reference is also made to the generalbackground art cited hereinabove, as well as to for example WO 94/29457,WO 96/34103 and WO 99/42077.

The immunoglobulin single variable domains, and polypeptides of theinvention can for example also be produced in the milk of transgenicmammals, for example in the milk of rabbits, cows, goats or sheep (seefor example U.S. Pat. No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S.Pat. No. 6,849,992 for general techniques for introducing transgenesinto mammals), in plants or parts of plants including but not limited totheir leaves, flowers, fruits, seed, roots or tubers (for example intobacco, maize, soybean or alfalfa) or in for example pupae of thesilkworm Bombyx mori.

Furthermore, the immunoglobulin single variable domains, andpolypeptides of the invention can also be expressed and/or produced incell-free expression systems, and suitable examples of such systems willbe clear to the skilled person. Some preferred, but non-limitingexamples include expression in the wheat germ system; in rabbitreticulocyte lysates; or in the E. coli Zubay system.

As mentioned above, one of the advantages of the use of immunoglobulinsingle variable domains is that the polypeptides based thereon can beprepared through expression in a suitable bacterial system, and suitablebacterial expression systems, vectors, host cells, regulatory elements,etc., will be clear to the skilled person, for example from thereferences cited above. It should however be noted that the invention inits broadest sense is not limited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thepolypeptides of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person,polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of immunoglobulin single variable domains orimmunoglobulin single variable domain-containing protein therapeuticsinclude strains of E. coli, Pichia pastoris, S. cerevisiae that aresuitable for large scale expression/production/fermentation, and inparticular for large scale pharmaceutical (i.e. GMP grade)expression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Richter Helm (Hamburg, Germany) or CMC Biologics (Soeborg, Denmark).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a immunoglobulin singlevariable domain-containing recombinant protein for which glycosylationis desired or required would necessitate the use of mammalian expressionhosts that have the ability to glycosylate the expressed protein. Inthis respect, it will be clear to the skilled person that theglycosylation pattern obtained (i.e. the kind, number and position ofresidues attached), will depend on the cell or cell line that is usedfor the expression. Preferably, either a human cell or cell line is used(i.e. leading to a protein that essentially has a human glycosylationpattern) or another mammalian cell line is used that can provide aglycosylation pattern that is essentially and/or functionally the sameas human glycosylation or at least mimics human glycosylation.Generally, prokaryotic hosts such as E. coli do not have the ability toglycosylate proteins, and the use of lower eukaryotes such as yeastusually leads to a glycosylation pattern that differs from humanglycosylation. Nevertheless, it should be understood that all theforegoing host cells and expression systems can be used in theinvention, depending on the desired polypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, thepolypeptide of the invention is glycosylated. According to anothernon-limiting aspect of the invention, the polypeptide of the inventionis non-glycosylated.

According to one preferred, but non-limiting aspect of the invention,the polypeptide of the invention is produced in a bacterial cell, inparticular a bacterial cell suitable for large scale pharmaceuticalproduction, such as cells of the strains mentioned above.

According to another preferred, but non-limiting aspect of theinvention, the polypeptide of the invention is produced in a yeast cell,in particular a yeast cell suitable for large scale pharmaceuticalproduction, such as cells of the species mentioned above.

According to yet another preferred, but non-limiting aspect of theinvention, the polypeptide of the invention is produced in a mammaliancell, in particular in a human cell or in a cell of a human cell line,and more in particular in a human cell or in a cell of a human cell linethat is suitable for large scale pharmaceutical production, such as thecell lines mentioned hereinabove.

As further described on pages 138 and 139 of WO 08/020,079, whenexpression in a host cell is used to produce the immunoglobulin singlevariable domains, and the polypeptides of the invention, theimmunoglobulin single variable domains, and polypeptides of theinvention can be produced either intracellularly (e.g., in the cytosol,in the periplasm or in inclusion bodies) and then isolated from the hostcells and optionally further purified; or can be producedextracellularly (e.g. in the medium in which the host cells arecultured) and then isolated from the culture medium and optionallyfurther purified. Thus, according to a non-limiting aspect of theinvention, the polypeptide of the invention is an amino acid sequence,polypeptide that has been produced intracellularly and that has beenisolated from the host cell, and in particular from a bacterial cell orfrom an inclusion body in a bacterial cell. According to anothernon-limiting aspect of the invention, the amino acid sequence, orpolypeptide of the invention is an amino acid sequence, or polypeptidethat has been produced extracellularly, and that has been isolated fromthe medium in which the host cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude those mentioned on pages 139 and 140 of WO 08/020,079.

Some preferred, but non-limiting secretory sequences for use with thesehost cells include those mentioned on page 140 of WO 08/020,079.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), a polypeptide of the invention (and in case of a hostorganism: in at least one cell, part, tissue or organ thereof). Theinvention also includes further generations, progeny and/or offspring ofthe host cell or host organism of the invention, which may for instancebe obtained by cell division or by sexual or asexual reproduction.

To produce/obtain expression of the immunoglobulin single variabledomains of the invention, the transformed host cell or transformed hostorganism may generally be kept, maintained and/or cultured underconditions such that the (desired) amino acid sequence, or polypeptideof the invention is expressed/produced. Suitable conditions will beclear to the skilled person and will usually depend upon the hostcell/host organism used, as well as on the regulatory elements thatcontrol the expression of the (relevant) nucleotide sequence of theinvention. Again, reference is made to the handbooks and patentapplications mentioned above in the paragraphs on the genetic constructsof the invention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theimmunoglobulin single variable domains of the invention may be expressedin a constitutive manner, in a transient manner, or only when suitablyinduced.

It will also be clear to the skilled person that the amino acidsequence, or polypeptide of the invention may (first) be generated in animmature form (as mentioned above), which may then be subjected topost-translational modification, depending on the host cell/hostorganism used. Also, the amino acid sequence, or polypeptide of theinvention may be glycosylated, again depending on the host cell/hostorganism used.

The amino acid sequence, or polypeptide of the invention may then beisolated from the host cell/host organism and/or from the medium inwhich said host cell or host organism was cultivated, using proteinisolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g. using aspecific, cleavable amino acid sequence fused with the amino acidsequence, or polypeptide of the invention) and/or preparativeimmunological techniques (i.e. using antibodies against the amino acidsequence to be isolated).

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference, in particular for theteaching that is referenced herein.

The invention will now be further described by means of the followingnon-limiting preferred aspects, figures and examples:

EXAMPLES Example 1 Identification of Anti-HGF ISVDs

1.1 Immunization and Selection of Neutralizing Anti-HGF Nanobodies

Two llamas (No. 085 and No. 092; Llama glama) were immunized with 100and 50 μg doses of human HGF (Peprotech, cat #100-39) according to thescheme outlined in Table 1. Proteins were administered in Stimuneadjuvant (Cedi Diagnostics, Lelystad, The Netherlands). Blood wascollected from these animals as indicated in Table 1.

TABLE 1 Immunization protocol Day Llama 085 Llama 092 Tissue collection100 ml naive blood (PBL_(N))  50 ml pre-immune blood (NC0) 0 100 μg  100μg   10 ml pre-immune blood 7 100 μg  100 μg  — 14 50 μg 50 μg — 21 50μg 50 μg — 28 50 μg 50 μg  10 ml immune blood 35 50 μg 50 μg — 39 150 mlimmune blood (PBL1) lymph node bow biopsy 43 150 ml immune blood (PBL2)50 50 μg 50 μg — 57 100 ml immune blood (NC1)

The animal experiments were conducted with the approval of the EthicalCommittee of the Faculty of Veterinary Medicine (University of Ghent,Belgium). Anti-HGF serum titers were evaluated using human HGF in anELISA type of method, essentially as described before (Roovers et al.,Cancer Immunol Immunother. 2007 56(3):303-317). The results are depictedin FIG. 1.

1.2 Library Construction

Anti-HGF Nanobodies were isolated using phage display, essentially asdescribed previously (Roovers et al., Cancer Immunol Immunother. 200756(3):303-317). Briefly: Peripheral blood mononuclear cells wereprepared from blood samples using Ficoll-Hypaque according to themanufacturer's instructions. Next, total RNA was extracted from thesecells and used as starting material for RT-PCR to amplify Nanobodiesencoding gene fragments. These fragments were cloned into phagemidvector pAX50. Phage was prepared according to standard methods (Rooverset al., Cancer Immunol Immunother. 2007 56(3):303-317.) and stored afterfilter sterilization at 4° C. for further use. Phage library size fromboth animals was 0.7×10⁸ and 2.1×10⁸, and percentages of insert 100 and91.3%, respectively.

Phage libraries from llama No. 085 and No. 092 were used for one roundof selection on human HGF. An overview of the different experimentalconditions used in different selection strategies are shown in Table 2.HGF antigen was either immobilized directly in a microtiter plate or wascaptured on a monoclonal antibody directed against the alpha or betachain of HGF. For direct immobilization HGF from R&D Systems (294-HG/CF;SEQ ID NO: 2) or Peprotech (100-39; SEQ ID NO: 3) was incubated in aNunc Maxisorp plate at concentrations between 50 nM and 0.5 nM. In thecapturing approach, different concentrations of HGF (from 5 nM to 0.05nM) were added to microtiter wells coated with either MAB294 (R&Dsystems) or sc-53301 (Santa Cruz). Bound phage was eluted by addition oftrypsin and rescued via infection of E. coli.

In another selection approach, phage was incubated in-solution withdifferent concentrations (5 nM to 0.5 nM) of biotinylated HGF(biotinylated at Ablynx according to standard procedures), captured onstreptavidin-coupled Dynabeads (Invitrogen) and eluted using trypsin.Phage eluates were rescued via infection of E. coli.

TABLE 2 Experimental conditions used in different selection strategiesMethod 1 Antigen 1 Elution 1 Method 3 Antigen 3 Elution 3 Passive plateHGF at 50 nM Trypsin streptavidin- Biotinylated Trypsin immobilizationcoupled HGF at 5 nM Dynabeads Passive plate HGF at 5 nM Trypsinstreptavidin- Biotinylated Trypsin immobilization coupled HGF at 0.5 nMDynabeads Passive plate HGF at 0.5 nM Trypsin streptavidin- BiotinylatedTrypsin immobilization coupled HGF at 0.05 nM Dynabeads Method 2 Antigen2 Elution 2 Passive plate Monoclonal Trypsin immobilization antibody at5 nM Passive plate Monoclonal Trypsin immobilization antibody at 0.5 nMPassive plate Monoclonal Trypsin immobilization antibody at 0.05 nM

Selection outputs were analyzed for enrichment factor (#4 phage presentin eluate relative to control). Based on these results the bestselections were chosen for further analysis. Individual colonies werepicked and grown in 96 deep well plates (1 ml volume). Nanobodyexpression was induced by addition of IPTG. Periplasmic extracts(volume: ^(˜)80 μl) were prepared according to standard methods (see forexample the prior art and applications filed by applicant cited herein)Nanobodies were expressed as tagged proteins containing both c-myc andHis6.

1.3 Selective Binding of the Nanobodies to Human HGF in ELISA

As a primary screen, Nanobody containing periplasmic extracts wereanalyzed for their ability to bind HGF. HGF from R&D Systems (referencenumber 294-HG/CF) was coated on ELISA plates at 2 μg/mL. Plates werewashed and subsequently blocked using PBS with 1% casein. Periplasmicextracts of individual clones, prediluted 1/10 in PBS/0.1% casein/0.05%Tween, were added and plates were incubated at RT for 2 hours. Bindingto immobilized HGF was detected using mouse anti-c-myc monoclonalantibody, followed by a horseradish peroxidase conjugatedrabbit-anti-mouse (human and bovine serum protein pre-absorbed)monoclonal antibody for detection. Individual clones were scored asputative HGF binders if the clones showed high optical densities in theassay. Overall, more than 90% of the clones were able to bind HGF (datanot shown).

1.4 Inhibition of Human HGF/c-Met-Fc Interaction in Alphascreen

Periplasmic extracts were then analyzed for their ability to block theinteraction of human HGF (R&D systems) with c-Met-Fc (R&D systems; SEQID NO: 5). To this end, an AlphaScreen™ assay (Perkin Elmer) was set upand used as a screening assay.

In brief, 5 μl of 1/5 prediluted periplasmic extract of individualNanobody clones were incubated with 3 nM biotinylated HGF, 2 nMc-Met-Fc, streptavidin coated donor beads and anti-human IgG1 FcNanobody covalently coupled AlphaScreen acceptor beads. Monoclonalantibody clone 24612 (R&D systems) known to inhibit the HGF/c-Met-Fcinteraction, was used as a positive control. Assays were read in anEnvision AlphaScreen™ option fitted multimode reader (Perkin Elmer).Individual clones were scored as putative HGF/c-Met-Fc interactioninhibiting if the presence of the periplasmic extract decreased thefluorescent signal of the acceptor beads. Both inhibitory andnon-inhibitory clones were identified. A 10-fold reduction in signalwith respect to the negative control (irrelevant periplasmic extract andbuffer) was used to select the HGF/c-Met inhibiting Nanobodies. As apositive control, anti-HGF monoclonal antibody (R&D systems, clone24612) was included. FIG. 2 depicts an example of screen of periplasmicextracts using AlphaScreen.

1.5 Off-Rate Determination of HGF Binding Nanobodies

Off-rate constants (K_(off)) of individual Nanobodies were determined bysurface plasmon resonance on a Biacore T100 instrument.

Human HGF (R&D) was amine-coupled to a CM5 sensor chip at a density of2500 relative units. Remaining reactive groups were inactivated usingethanolamine. Nanobody binding was assessed at a single dilution ofperiplasmic extract, whereas c-Met was tested with a singleconcentration of 10 nM. Each sample was injected for 2 min at a flowrate of 45 μl/min to allow for binding to chip-bound antigen. Next,binding buffer without Nanobody was sent over the chip at the same flowrate to allow dissociation of bound Nanobody. After 10 min, remainingbound analyte was removed by injecting regeneration solution (1M NaCl,50 mM NaOH). Binding/dissociation curves were used to calculate k_(off)values, which are shown in Table 3.

TABLE 3 Off-rate screening of unpurified Nanobodies using BIAcore Clonekd (s⁻¹) Clone kd (s⁻¹) Clone kd (s⁻¹) 1A1 1.89E−02 2A1 4.51E−03 6F65.45E−03 1A7 3.21E−03 2A3 2.19E−03 6G6 1.78E−03 1B4 3.13E−03 2A42.62E−03 6H6 1.39E−02 1B9 1.05E−02 2A9 3.27E−03 6B6 9.37E−06 1B112.82E−03 2B3 3.14E−03 5B7 1.75E−03 1C6 6.50E−03 2B7 3.26E−03 5C71.90E−03 1C7 2.86E−03 2B9 3.20E−03 5E8 1.63E−03 1C8 3.25E−03 2B111.78E−03 5G9 8.00E−04 1D7 3.41E−03 2C1 2.18E−03 5D10 1.99E−03 1D83.36E−03 2C3 3.31E−03 6B2 7.74E−04 1D10 3.42E−03 2C5 2.56E−03 6E103.24E−04 1E2 3.12E−03 2C9 3.28E−03 6E11 1.68E−03 1E4 3.63E−03 2C112.68E−03 8G3 1.95E−03 1E7 3.49E−03 2D2 2.62E−03 8C10 8.35E−05 1E83.61E−03 2E7 3.23E−03 9E3 1.72E−03 1E9 3.14E−03 2F5 2.15E−03 9B51.43E−03 1E11 3.24E−03 2G8 2.32E−03 9H5 1.55E−03 1F3 3.45E−03 2H83.29E−03 9E6 2.18E−03 1F6 6.57E−03 2H11 3.11E−03 9G6 1.07E−03 1F73.36E−03 2F11 3.15E−03 9A10 2.61E−03 1F8 3.45E−03 6A6 1.64E−03 10G33.77E−04 1F10 1.08E−02 7G6 9.36E−04 c-Met 5.09E−03 1G1 3.44E−03 6C62.75E−02 c-Met 4.38E−03 1G5 3.78E−03 6D6 2.30E−04 1H8 3.37E−03 6E61.70E−03

1.6 HGF-Inhibiting Nanobody Expression and Purification

12 inhibitors of the HGF/c-Met interaction were selected for furthercharacterization. The aligned sequences are given in FIG. 3. SelectedNanobody inhibitors were expressed in the periplasmic space of E. colias c-myc, His6-tagged proteins in a culture volume of 50 mL. Expressionwas induced by addition of 1 mM IPTG and allowed to continue for 4 h at37° C. After spinning the cell cultures, periplasmic extracts wereprepared by freeze-thawing the pellets. These extracts were used asstarting material for immobilized metal affinity chromatography (IMAC).Nanobodies were eluted from the column with 250 mM imidazole andsubsequently subjected to gel filtration to PBS. The purified Nanobodyproteins were subsequently analyzed for purity and molecular weight onCoomassie-stained SDS-PAGE gels, run under reducing conditions (data notshown).

1.7 Affinity Determination of Nanobodies Binding HGF

Affinity constants (K_(d)) of individual purified Nanobody clones weredetermined by surface plasmon resonance on a Biacore T100 instrumentessentially as described above. Kd, ka and kd values for HGF binding ofselected Nanobody clones are summarized in Table 4. The average wastaken of an analysis performed on R&D material and Peprotech material.

TABLE 4 kinetic and affinity constants of anti-HGF Nanobodies Clone ka(1/Ms) kd (1/s) Kd (nM) 1E2 7.52 (±0.55) * 10⁴ 1.00 (±0.52) * 10⁻⁴ 1.36± 0.78 6E10 4.99 (±0.55) * 10⁴ 5.50 (±7.07) * 10⁻⁵ 1.14 ± 1.59 6B2 2.78(±0.45) * 10⁴ 2.15 (±0.30) * 10⁻⁶ 0.08 ± 0.02 1E9 9.20 (±5.95) * 10⁴5.20 (±0.04) * 10⁻⁴ 7.16 ± 4.59 6G6 7.53 (±0.66) * 10⁵ 5.73 (±4.88) *10⁻³ 7.37 ± 5.85 7G6 1.06 (±0.24) * 10⁶ 6.91 (±0.67) * 10⁻⁴ 0.67 ± 0.09

The dissociation curves of some of the clones were bi-phasic. Thereforethe dissociation curves were split into 2 parts ranging from 125 to 185seconds and 300 to 720 seconds after the injection start. The fittedoff-rates are respectively kd1 and kd2 and the corresponding values aregiven in Table 5.

TABLE 5 off-rates of anti-HGF Nanobodies which showed a heterogeneousdissociation profile Clone kd1 (1/s) kd2 (1/s) 9H5 2.41 (±1.36) * 10⁻³9.52 (±8.60) * 10⁻⁴ 9G6 2.37 (±1.32) * 10⁻³ 1.02 (±0.29) * 10⁻³ 5C7 6.05(±0.35) * 10⁻³ 8.21 (±0.64) * 10⁻⁴ 6H6 3.13 (±0.39) * 10⁻³ 3.32(±0.70) * 10⁻³ 6F6 5.77 (±0.30) * 10⁻³ 2.13 (±0.23) * 10⁻³

1.8 Inhibition of Human HGF/c-Met-Fc Interaction in AlphaScreen™)

Serial dilutions of purified Nanobodies were then analyzed for theirability to block the interaction of human HGF with c-Met-Fc using thesame AlphaScreen™ assay as described above (1.4). Representative dataare shown in FIG. 4. At last 8 Nanobodies have been identified whichinhibit the HGF/c-Met interaction with IC₅₀ values ranging from 1.8nM-26.7 nM and bind to HGF with an affinity constant ranging from about100 pM up to about 10 nM. Table 6 gives a summary of the obtainedresults on these 8 anti-HGF Nanobodies.

TABLE 6 Summary of obtained results on several anti-HGF candidates.Clone IC50 (nM) K_(d) (nM) kd (s⁻¹) 1E2 1.8 1.36 ± 0.78 1 × 10e−4 9H52.1 heterogeneous 1 × 10e−3 9G6 6.6 heterogeneous 1 × 10e−3 6E10 3.01.14 ± 1.59 5 × 10e−5 6B2 2.8 0.08 ± 0.02 1 × 10e−5 6H6 26.7heterogeneous 3 × 10e−3 1E9 4.5 7.16 ± 4.59 5 × 10e−4 6G6 2.8 7.37 ±5.85 6 × 10e−3

1.9 Formatting Nanobodies of the Invention

To test whether selected Nanobodies have potential as anticancer agents,a strategy to increase the serum half-life is preferred (as for exampledescribed in patent application WO 04/041865), since the serum half-lifeof a mono- or bivalent Nanobody (approximately 15 or 30 KDa,respectively) is not optimal for this therapeutic indication.

Human serum albumin specific Nanobody ALB, cross reactive with mouseserum albumin, was chosen. Here we describe the construction ofbispecific Nanobodies consisting of an anti-HGF Nanobody and a serumalbumin binding immunoglobulin single variable domain (ALB), allseparated by a 9 (GS) amino acid linker peptide and resulting inconstructs 1E2-9GS-ALB and 6E10-9GS-ALB.

Cloning was performed such that said Nanobodies are translationallyfused at their C-terminus to an anti-human serum albumin (HSA) bindingNanobody (ALB), separated by a 9GS-linker (amino acid sequence GGGGSGGGS(SEQ ID NO:119). The constructs have an additional C-terminal 3×FLAG andHis₆-tag (SEQ ID NO: 116).

1.10 Trispecific Nanobodies of the Invention

Nanobodies 1E2-9GS-ALB-35GS-αEGFR, αEGFR-9GS-ALB-35GS-1E2,6E10-9GS-ALB-35GS-αEGFR and αEGFR-9GS-ALB-35GS-6E10 are cloned into thesame expression plasmid and fused to the same ALB Nanobody as describedin Example 1.9, but such that the HGF binding Nanobodies aretranslationally fused at either their C-terminus or N-terminus to αEGFRNanobodies, separated by the Alb8

Nanobody and a 35GS-linker. The αEGFR Nanobodies are described in WO05/044858, WO 04/041867 and/or WO07/042,289. As above, these constructscarry C-terminal 3×FLAG and His₆-tags.

Nanobodies 1E2-9GS-ALB-35GS-αVEGF, αVEGF-9GS-ALB-35GS-1E2,6E10-9GS-ALB-35GS-αVEGF and αVEGF-9GS-ALB-35GS-6E10 are cloned into thesame expression plasmid and fused to the same ALB Nanobody as describedin Example 1.9, but such that the HGF binding Nanobodies aretranslationally fused at either their N-terminus or C-terminus to αVEGFNanobodies, separated by the ALB Nanobody and a 35GS-linker. The αVEGFNanobodies are described in WO 08/101,985. As above, these constructscarry C-terminal 3×FLAG and His₆-tags.

Example 2 α-HGF ISVDs in Tumor Therapy: Materials and Methods

2.1 Materials

All reagents where purchased from Sigma-Aldrich (St. Louis, Mo.) unlessotherwise stated. With respect to the preparation of the ⁸⁹Zr-labeledNanobody constructs, no special measures were taken regarding workingunder strict metal-free conditions. Df-Bz-NCS was purchased fromMacrocyclics (cat. No. B705). [⁸⁹Zr]Zr-oxalate in 1.0 M oxalic acid 0.15GBq/nmol) was from IBA molecular (cf. www.iba.be/molecular).

2.2 Cell Line

The glioblastoma cell line U87 MG, which contains an autocrine HGF-loop,was obtained from the American Type Culture Collection (www.ATCC.com)and cultured in DMEM supplemented with 5% FBS. All cells were culturedat 37° C. with 5% CO₂.

2.3 Preparation of ⁸⁹Zr-1E2-ALB and ⁸⁹Zr-6E10-ALB

For preparation of the ⁸⁹Zr conjugates, the ⁸⁹Zr was coupled to theNanobody by use of the bifunctional chelate p-isothiocyanatobenzyldesferrioxamine (Df-Bz-NCS), essentially as described by Vosjan et al.,“Conjugation and radiolabeling of monoclonal antibodies withzirconium-89 for PET imaging using the bifunctional chelatep-isothiocyanatobenzyl-desferrioxamine” (Vosjan M J, Perk L R, Visser GW, Budde M, Jurek P, Kiefer G E, van Dongen G A. Nat Protoc. 2010;5(4):739-43).

In short, 1-2 mg αHGF-Nanobody was premodified with a 3-fold molarexcess of Df-Bz-NCS. After PD-10 column purification the premodifiedNanobody was labeled with ⁸⁹Zr (37 MBq) in 0.25 M HEPES buffer pH 7.0 atroom temperature in a total volume of 2 mL. The ⁸⁹Zr-Df-Bz-NCS-Nanobodywas purified by PD-10 column using 0.25 M NaOAc with 5 mg mL⁻¹ gentisicacid, pH 5.5, as eluent.

2.4 Quality Control of ⁸⁹Zr-1E2-ALB and ⁸⁹Zr-6E10-ALB

All radioactive conjugates were analyzed by instant thin layerchromatography (ITLC) to determine the labeling efficiency andradiochemical purity. The integrity of the Nanobody was analyzed by highperformance liquid chromatography (HPLC) and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed byphosphor imaging (Storm820, GE Healthcare). Immunoreactivity wasdetermined by a HGF-coated enzyme-linked immunosorbent assay essentiallyas described by Collingridge (Collingridge et al., “The development of[(124)I]iodinated-VG76e: a novel tracer for imaging vascular endothelialgrowth factor in vivo using positron emission tomography.” Cancer Res2002; 20:5912-9).

2.5 Biodistribution Study

The distribution of ⁸⁹Zr-labeled αHGF-Nanobodies was examined in nudemice (HSD: Athymic Nude-Foxn1^(nu), 20-30 g; Harlan Laboratories, Horst,The Netherlands) inoculated subcutaneously with 2×10⁶ U87 MG cells attwo lateral sides. All animal experiments were done according to NIHPrinciples of Laboratory Animal Care and Dutch national law (“Wet op dedierproeven”, Stb 1985, 336).

Mice bearing U87 MG xenografts (size ^(˜)100 mm³) were injected with0.37 MBq ⁸⁹Zr-Df-Bz-NCS-1E2-ALB or 0.37 MBq ⁸⁹Zr-Df-Bz-NCS-6E10-ALB viathe retro-orbital plexus. Unlabeled Nanobody was added to the injectionmixture to obtain a final dose of 30 μg per mouse. At 1, 2, 3 or 7 dayspost injection (pi) five mice per group were anesthetized, bled, killedand dissected. Blood, tumor and normal tissues were weighed andradioactivity was measured in a gamma counter (Wallac, Turku, Finland).Radioactivity uptake for each sample was calculated as the percentage ofthe injected dose per gram of tissue (% ID/g).

In addition, a Nanobody dose-diminishing study was performed. To thisend, 5, 10, 20 and 30 μg of ⁸⁹Zr-labeled 1E2-ALB (0.23-0.83 MBq) wasinjected in mice bearing U87 MG xenografts, at 3 days p.i. 5 mice pergroup were examined as described above.

2.6 Blood Kinetics in Mice

Blood concentrations of αHGF-Nanobodies were examined in two groups oftwo mice. One group of tumor-bearing mice received 0.37 MBq ⁸⁹Zr-1E2-ALB(30 while the other group received 0.37 MBq ⁸⁹Zr-6E10-ALB (30 Blood wascollected at 1 and 3 h, and at 1, 2, 3 and 7 days pi by tail lacerationand radioactivity was measured in a gamma counter. Radioactivity foreach sample was calculated as the percentage of the injected dose pergram of blood (% ID/g).

2.7 Therapy Study

The therapeutic effectiveness of the αHGF-Nanobodies was studied in thesame nude mice model as described for the biodistribution study.

To this end, 7 groups of 6 mice with established U87 MG xenografts wereevaluated. At the start of this study mean tumor size was ^(˜)100 mm³,and was similar for the different treatment groups. All mice receivedi.p. treatment 3 times a week for 5 weeks. Group 1 was the control groupand received 200 μl of saline solution per dose. Group 2, 3 and 4,received 10, 30 and 100 μg of Nanobody 1E2-ALB, respectively. Group 5, 6and 7 received 10, 30 and 100 μg of Nanobody 6E10-ALB, respectively.Body weight and tumor volume were measured 3 times a week up to 70 daysafter end of treatment.

2.8 Statistical Analysis

Biodistribution and therapy experiments were statistically analyzedusing SPSS 15.0 software. Differences in tissue uptake between injectedconjugates as well as differences in average tumor volume between thevarious treatment groups were statistically analyzed for each differenttime point using Student t-test for unpaired data. Survival wascalculated using Kaplan-Meier curves. Two-sided significance levels werecalculated and P<0.05 was considered statistically significant.

Example 3 Radiolabeling and Quality Control of ⁸⁹Zr-1E2-ALB and⁸⁹Zr-6E10-ALB

Labeling of both Nanobodies with ⁸⁹Zr resulted in overall labelingyields of 75-90%, after PD-10 column purification. Radiochemical puritywas always >97% as determined with ITLC and confirmed with HPLC.Integrity of the Nanobodies was optimal as determined by HPLC andSDS-PAGE. Immunoreactivity of ⁸⁹Zr-1E2-ALB and ⁸⁹Zr-6E10-ALB wasdetermined by HGF-coated ELISA and was similar to that of the reference¹³¹I-labeled αHGF-Nanobodies (^(˜)50%).

Example 4 Biodistribution Study

For biodistribution, studies nude mice bearing U87 MG xenografts wereinjected with either 0.39±0.01 MBq ⁸⁹Zr-1E2-ALB or 0.37±0.01 MBq⁸⁹Zr-6E10-ALB. Biodistribution at 1, 2, 3, or 7 days p.i. is shown inFIG. 5. Both α-HGF-Nanobodies showed similar biodistributions withselective tumor uptake; no significant differences were observed(P>0.01). While blood levels gradually decreased over time, tumor uptakeremained relatively stable. Blood levels were 12.6±0.7, 7.2±1.0,3.4±0.3, and 0.3±0.1% ID/g for ⁸⁹Zr-1E2-ALB and 13.1±0.6, 7.4±0.6,3.5±0.5, and 0.5±0.1% ID/g for ⁸⁹Zr-6E10-ALB at 1, 2, 3, and 7 daysp.i., respectively. Tumor uptake at these time points was 7.8±1.1,8.9±1.0, 8.7±1.5, and 7.2±1.6% ID/g for ⁸⁹Zr-1E2-ALB, and 7.5±0.8,8.8±1.3, 6.5±2.5, and 6.3±4.0% ID/g at 1, 2, 3, and 7 days p.i.,respectively for ⁸⁹Zr-6E10-ALB. Tumor uptake was higher than in normalorgans, except for kidneys. The latter is typical for small proteins,which are rapidly cleared via the kidneys.

Example 5 Dose-Diminishing Study

A dose-diminishing study was performed with ⁸⁹Zr-1E2-ALB to determinethe optimal Nanobody dose for in vivo imaging. Nude mice bearing U87 MGxenografts were injected with 0.32±0.01, 0.47±0.01, 0.47±0.01, or0.83±0.01 MBq ⁸⁹Zr-1E2-ALB, containing 5, 10, 20 or 30 μg 1E2-ALB,respectively. Three days p.i. similar biodistribution was seen for alldose groups (FIG. 6). No significant differences were observed in tumoruptake, being 8.2±1.2, 8.1±1.3, 6.3±1.7, 6.9±1.1% ID/g for the 5, 10,20, and 30 μg dose groups, respectively. High uptake in kidneys wasobserved for all dose groups. Also no significant differences wereobserved between the different dose groups (P>0.01).

Example 6 Blood Kinetics in Mice

Blood kinetics of 0.39±0.01 MBq ⁸⁹Zr-1E2-ALB (30 μg) and 0.37±0.01 MBq⁸⁹Zr-6E10-ALB (30 μg) appeared to be similar (FIG. 7). Blood levels ofNanobody constructs were 41.7±0.6 and 35.1±1.48% ID/g 1 h afterinjection, for ⁸⁹Zr-1E2-ALB and ⁸⁹Zr-6E10-ALB, respectively. This slowlydecreased from 4.3±0.1 to 0.3±0.1% ID/g between 72 and 168 h p.i. for⁸⁹Zr-1E2-ALB and from 4.2±0.1 to 0.5±0.1% ID/g for ⁸⁹Zr-6E10-ALB.

Example 7 Therapy Study

Mice who received Nanobodies showed tumor growth delay in comparison tothe control PBS-group (group 1) (FIG. 8A). Within the treatment scheduleof 35 days all control mice were sacrificed due to the large volumes ofthe tumors. Mice who received α-HGF-Nanobodies responded to treatment;mice who received the lowest dose (group 2 and 5; 10 μg) had minimalbenefit while the highest dose groups (group 4 and 7; 100 μg) showedsignificant tumor growth delay with 4 out of 6 mice (7 out of 11 tumors)being cured in group 4 (1E2-ALB), and 3 out of 6 mice (6 out of 11tumors) being cured in group 7 (6E10-ALB). In the intermediate dosegroups, no cures were observed in group 6 (6E10-ALB), while 3 out of 6mice (5 out of 11 tumors) were cured in group 3 (1E2-ALB) (FIG. 8B).

At the end of treatment (day 35), only mice in the intermediate andhighest dose groups were alive, and followed till day 108 after start oftreatment. At the end of the study 4 out of 6 mice (66%) were cured ingroup 4 (100 μg 1E2-ALB), and 3 out of 6 mice (50%) in group 7 (100 μg6E10-ALB), while 2 out of 6 mice (33%) were cured in group 3 (30 μg1E2-ALB). In contrast, all mice in group 5 (30 μg 6E10-ALB) facedre-growth of tumors during follow up (FIG. 8C).

Example 8 Comparison with Contemporaneous Anti-HGF Antibodies

In 2001, Cao et al. (Proc Natl Acad Sci USA 2001; 98:7443-8) reportedthe first in vivo results with monoclonal antibodies that bind to HGF. Amixture of 3 antibodies (200 μg/mouse every day until day 20) wasinjected in mice, which were injected one day before start of therapywith C-127 cells or U118 cells. All mice showed growth inhibition ascompared to the control groups. In an established U118 tumor model, micereceived a mixture of A-1 and A-7 3 times a week for 10 weeks, tumorgrowth delay was seen. Cao et al. needed a combination of 3 monoclonalantibodies to achieve neutralizing activity to HGF in glioma xenografttumors, and suggested that the complex heterodimeric structure of HGFmakes it necessary to simultaneously target multiple HGF epitopes bycombining mAbs. Moreover, no cures were observed.

AMG102 (rilotumumab; Amgen, Inc.) was identified in an extensive screen,resulting in 3 potential candidates, of which each recognized adifferent epitope. Although AMG102 had intermediate affinity for HGF [asjudged by binding affinity], it was the only mAb identified thatcompletely blocked the binding of HGF to c-Met (Kim et al. 2006 ClinCancer Res 12:1292-1298). Nevertheless, Schöffski and colleaguesdemonstrated that no significant growth inhibition occurred with AMG102in metastatic renal cell carcinoma (Schöffski et al. 2010 BJU Intdoi:10.1111/j.1464). Similarly, HGF and its receptor c-Met have beenimplicated in the pathogenesis of glioblastoma (GBM), but Wen andcolleagues showed in a phase II study that AMG102 monotherapy treatmentat doses up to 20 mg/kg was not associated with significant antitumoractivity in the selected patient groups (Wen et al. 2011 Neuro-Oncologydoi:10.1093/neuonc/noq198). Hence, merely blocking the binding of HGF toc-Met does not warrant tumor inhibition.

The monoclonal antibody AV-299 (also known as SCH900105), which isdeveloped by Aveo pharmaceuticals/Schering Plough, is not published inany peer reviewed journal but only discussed on posters. No substantiveinformation is available of AV-299, such as its sequence. Nevertheless,the information on these posters is not consistent.

It can be concluded that the monovalent Nanobodies of the presentinvention outperform the contemporaneous monoclonal antibodies, whichare bivalent.

Example 9 In Vitro Efficacy of Nanobodies 1E2 and 6E10 AgainstHGF-Driven Proliferation in Multiple Myeloma Cell Lines

The in vitro efficacy of Nanobodies 1E2 and 6E10 on HGF inducedproliferation is assessed in c-Met positive human multiple myelomacells. Both HGF autocrine (ANBL-6) as well as paracrine (INA-6, 1H-1 andOH-2) multiple myeloma cell lines are analyzed according to Hov et al.(Hov et al. 2004; Clin Cancer Res 10, 6686-6694; and Hov et al., 2009;Eur J Haematology 82, 277-287).

Example 10 Analysis of Efficacy of Trispecific HGF/EGFR Nanobodies onPI3K Signaling

The c-Met as well as the EGFR can signal via the PI3K pathway whichconveys mitogenic signals. To demonstrate simultaneous targeting of theEGFR and c-Met receptor phosphorylation of AKT, a downstream target inthe PI3K pathway, can be monitored. To this end, unstimulated cells,cells treated with EGF or HGF or cells treated with both cytokines arein parallel incubated with unspecific, parental control or bispecificNanobodies, Nanobodies 1E2 and 6E10 are each coupled to Nanobodiesinhibiting EGFR as described in Example 1.10. Alternatively, one canalso assess cells which overexpress EGFR and/or have an autocrine HGFloop which activates c-Met signaling. AKT is a major downstreamsignaling component of the PI3K pathway and phosphorylation of thisprotein is a key indicator of signaling via this pathway.

Example 11 Analysis of Efficacy of Trispecific HGF/EGFR Nanobodies onMAPK Signaling

EGFR and c-Met receptor can signal via the MAPK pathway. To demonstratetargeting of the EGFR and c-Met receptor, phosphorylation of ERK1/2, amajor downstream target in the MAPK pathway, can be monitored. To thisend, unstimulated cells, cells treated with EGF or HGF or cells treatedwith both cytokines are in parallel incubated with unspecific,monospecific, or bispecific Nanobodies essentially according to Example1.10. Alternatively, one can also assess cells which overexpress EGFRand/or have an autocrine HGF loop which activates c-Met signaling.

Example 12 Analysis of Efficacy of Trispecific HGF/EGFR Nanobodies onInhibiting Proliferation

A431 cells display high cell surface levels of EGFR and medium high cellsurface expression of c-Met as was independently confirmed in othersstudies.

Inhibition of A431 proliferation by bispecific HGF/EGFR Nanobodiesessentially according to Example 1.10 can be measured in CellTiterGlow™assay after 48 hours.

Example 13 In Vitro Analysis of Migration of Cells after Treatment withTrispecific Nanobodies

Active c-Met signaling is involved in cell migration and invasion.Efficacy of the trispecific Nanobody can be determined by measuringinhibition of HGF-induced migration. For this purpose, the HGF-induciblecell line A549 is treated with HGF in the presence or absence of thebispecific Nanobody, monospecific Nanobodies against HGF and inhibitorsof EGFR. Alternatively, the migration of cells through an 8 μm pore ismeasured in a time dependent manner on an Acea Real Time analyzer usingCIM-plates as a read out.

Example 14 Analysis of Efficacy of Trispecific HGF/VEGF Nanobodies in aKP4 Pancreatic Xenograft Tumor Model

KP4 cells are cultured in growth media that consists of RPMI 1640 media(Invitrogen), 2 mM L-glutamine, and 10% fetal bovine serum. To preparecells for inoculation into mice, cells are trypsinized and subsequentlywashed with ten milliliters of sterile IX phosphate buffered saline(PBS). A subset of cells is counted by trypan blue exclusion and theremainder of cells is resuspended in 100 μl of sterile 1×PBS to aconcentration of 5×10⁷ cells per milliliter. Mice are inoculatedsubcutaneously in the right sub-scapular region with 5×10⁶ KP4 cells.Tumors are monitored until they reach a mean volume of 230 mm.

Mice are randomized into 5 groups of ten mice each and treatment isinitiated. Mice in Group 1 are treated with monospecific HGF Nanobody.Mice in Group 2 are treated with monospecific αVEGF Nanobody. Mice inGroup 3 are treated with a trispecific HGF/αVEGF Nanobody essentiallyaccording to Example 1.10. Mice in Group 4 are treated with amonospecific αVEGF Nanobody as well as a monospecific HGF Nanobody. Micein Group 5 are treated with a negative control (unrelated Nanobody).Tumor volumes are measured twice per week and animals are monitored for25 days.

Example 15 Analysis of Efficacy of Trispecific HGF/αVEGF Nanobodies in aNSCLC Xenograft Tumor Model

Human NSCLC cells (A549, DSMZ, Braunschweig, Germany) are cultured ingrowth media that consists of RPMI 1640 media (Invitrogen), 2 mML-glutamine, and 10% fetal bovine serum. To prepare cells forinoculation into mice, cells are trypsinized and subsequently washedwith ten milliliters of sterile IX phosphate buffered saline (PBS). Asubset of cells is counted by trypan blue exclusion and the remainder ofcells is resuspended in 100 μl of sterile IX PBS to a concentration of5×10⁷ cells per milliliter. Mice are inoculated subcutaneously in theright sub-scapular region with 5×10⁶ human A549 cells. Tumors aremonitored until they reach a mean volume of 200 mm.

Mice are randomized into 5 groups of ten mice each and treatment isinitiated. Mice in Group 1 are treated with monospecific HGF Nanobodyaccording to the invention. Mice in Group 2 are treated withmonospecific αVEGF Nanobody. Mice in Group 3 are treated with atrispecific HGF/αVEGF Nanobody essentially according to Example 1.10.Mice in Group 4 are treated with a monospecific αVEGF Nanobody as wellas a monospecific HGF Nanobody according to the invention. Mice in Group5 are treated with a negative control (unrelated Nanobody). Tumorvolumes are measured twice per week and animals are monitored for 25days.

Example 16 Analysis of Efficacy of Tetraspecific HGF/VEGF/EGFRNanobodies in a NSCLC Xenograft Tumor Model

Human NSCLC cells (A549, DSMZ, Braunschweig, Germany) are cultured ingrowth media that consists of RPMI 1640 media (Invitrogen), 2 mML-glutamine, and 10% fetal bovine serum. To prepare cells forinoculation into mice, cells are trypsinized and subsequently washedwith ten milliliters of sterile IX phosphate buffered saline (PBS). Asubset of cells is counted by trypan blue exclusion and the remainder ofcells is resuspended in 100 of sterile 1×PBS to a concentration of 5×10⁷cells per milliliter. Mice are inoculated subcutaneously in the rightsub-scapular region with 5×10⁶ human A549 cells. Tumors are monitoreduntil they reach a mean volume of 200 mm.

Mice are randomized into 6 groups of ten mice each and treatment isinitiated. Mice in Group 1 are treated with a monospecific HGF Nanobodyaccording to the invention. Mice in Group 2 are treated withmonospecific αVEGF Nanobody. Mice in Group 3 are treated with amonospecific αEGFR Nanobody. Mice in Group 4 are treated with amonospecific αVEGF Nanobody, a monospecific αEGFR Nanobody as well as amonospecific HGF Nanobody according to the invention. Mice in Group 5are treated with a tetraspecific HGF/αVEGF/αEGFR/ALB Nanobody. Mice inGroup 6 are treated with a negative control (unrelated Nanobody). Tumorvolumes are measured twice per week and animals are monitored for 25days.

Sequence Tables:

TABLE B-1 Prior art sequences Name SEQ ID NO Amino acid sequenceshuman HGF 1 MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKT (hHGF)TLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIHRHKFLPERYPDKGFDDETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQSNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLGKGRSYKGTVRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDL QENYCRNP rec-hHGF 2QRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKG (Peprotech)LPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYK VPQS rec hHGF 3QRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKG (R&D systems)LPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAEGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS Human c-Met 4MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTA or hc-MetETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFTGLIAGVVSISTALLLLLGFFLWLKKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSSQNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHVVIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS recombinant 5ECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYV humanLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMAL c-Met/FcVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEE chimeraPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYPDDHPLHSISV (R&D systems)RRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFTHIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH Alb11 114  EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC TIGGSLSRSSQGTLVTVSSAlb8 115   EVQLVESGGGLVQPGNSLRLSCALSGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSAAAEQKLISEEDLNGAAHHHHHH Tag-1 or 3xFLAG- 116  GAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH His₆

TABLE B-2Sequences for CDRs and frameworks, plus preferred combinations as provided in forformula I, i.e. FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (the following terms: “ID”refersto the given SEQ ID NO. Preferred combinations of FR and CDR sequences for eachNanobody construct are used interchangeably throughout the application)Clone* ID FR1 ID CDR1 ID FR2 ID CDR2 1E2:  7 EVQLVESGGGLVQPG 28 SYAMG 40WFRQAPG 52 GISWSGSSS GSLRLSCAASGRTFS KEREFVA YYADSVKG 1E9:  8EVQLVESGGGLVQPG 29 HYTIG 41 WFRQAPG 53 CISSLDGST GSLRLSCAASGFTLD NEREGVSYYADSVKG 5C7: 10 EVQLVESGGGLVQAG 30 SSHMA 42 WFRQVPE 54 AISRSGGTTDSLRLSCAASGGTFG KEREFVA YYADSVKG 6B2: 15 EVQLVESGGGLVQAG 31 SYAMG 43WFRQAPG 55 AINWSGDST GSLRLSCAASGRTFS KEREFVA YYADSVKG 6B6: 16EVQLVESGGGLVQPG 32 DYAMS 44 WGRQAPG 56 AISWNGGST GSLRSLCAASGFTFD KGLEWVSYYAESMKG 6E10: 18 EVQLVESGGGLVQAG 33 SYAMG 45 WFRQAPG 57 AISWSGGSTGSLRLSCAASGRTDS KEHEFVA YYADSVKG 6F6: 20 EVQLVESGGGLVQPG 34 INAMG 46WYRQAPG 58 VIN-SGGST GSLRLSCAASGSILG KQRELVA NYADSVKG 6G6: 21EVQLVESGGGLAQAG 35 NYAMG 47 WFRQAPG 59 AISRSGSIT GSLRLSCAASGRSLS KEREIVCTYADSVKG 6H6: 22 EVQLMESGGGLVQAG 36 SYAMG 48 WFRQAPG 60 AISSSGGYTGSLRLSCAASGRTFS KERENVA YYPDSVKG 7G6: 23 EVQLVESGGGLVQAG 37 PYTMG 49WFRRAPG 61 ARTWSGGVA GSLRLSCAVSGRTFS KEREFVA WYADSVKG 9G6: 24EVQLVESGGGLVQAG 38 DYTMG 50 WFRQAPG 62 RINTGGPIT GSLRLSCAASGRTLS KEREFVASYSDSVKG 9H5: 25 EVQLVESGGGLVQPG 39 LNPMG 51 WYRQAPG 63 TVT-GEGRTGSLRLSCAASGSIFN KQRELVA NYSDSVKG Clone* ID FR3 ID CDR3 ID FR4 ID 1E2: 64RFTISRDNAKNTVYLQ 76 DPVGRADLYEYDY 88 WGQGTQVTVSS 100 MNSLKPEDTAVYYCAA1E9: 65 RFTISRDNAKDTVYLQ 77 AGCGAYGLIPYDY 89 WGQGTQVTVSS 101MNSLKPEDTAVYYCAV 5C7: 66 RCTISRDNAKNTVYLQ 78 ARFWGSTSTRMDDYQY 90WGQGTQVTVSS 102 MNSLKPEDTAVYYCAA 6B2: 67 RFTISRDNAKNTVYLQ 79GDVGRPDLYEYDY 91 WGQGTQVTVSS 103 MNSLKPEDTAVYYCAA 6B6: 68RFTVSRDNAKNTLYLQ 80 ALDPLGVLAGTSGIYDY 92 WGQGTQVTVSS 104MNSLKSEDTAVYYCAK 6E10: 69 RFTISRDNAKNTLYLQ 81 SYRWGITHEYEY 93WGQGTQVTVSS 105 MNSLKPEDTAVYYCGA 6F6: 70 RFTISRDNAKNTVYLQ 82DEWGDYPGQVYDY 94 WGQGTQVTVSS 106 MNSLKPEDTAVYYCNA 6G6: 71RFTIAKDNAANTVYLQ 83 DPMYYGIPDQNWDY 95 WGQGTQVTVSS 107 MNSLKPEDTAAYYCAA6H6: 72 RFTISRDTAKNTVYLQ 84 TQPNFGWQLLLLQTEYDY 96 WGQGTQVTVSS 108MNSLKPDDTAVYYCAG 7G6: 73 RFTISSDNAENMVYLQ 85 KSPGRTYSPREERAYAR 97WGQGTQVTVSS 109 MNSLKPEDTAVYYCAA 9G6: 74 RFTISRDNAKNTVYLQ 86RLPTKMSPRDYSSYAY 98 WGQGTQVTVSS 110 MNSLKPEDTAVYNCAA 9H5: 75RFTISRDNAKNTVYLQ 87 AFWAYDDAY 99 WGQGTQVTVSS 111 MNSLKPEDTAVYYCNA

TABLE B-3 Amino acid sequences of immunoglobulinsingle variable sequences of the invention Name of clone SEQ ID NO:Amino acid sequences 1C7:  6 EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAGISWSGGSTYYADSVKGRFTISRDNAKNTVYLRMNSLKPEDTAVYYCAADPVGRADLYEYDYWGQGTQVTVSS 1E2:  7EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAGISWSGSSSYYADSVKGRFTISRDNAKNYVYLQMNSLKPEDTAVYYCAADPVGRADLYEYDYWGQGTQVTVSS 1E9:  8EVQLVESGGGLVQPGGSLRLSCAASGFTLDHYTIGWFRQAPGNEREGVSCISSLDGSTYYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAVAGCGAYGLIPYDYWGQGTQVTVSS 5B7:  9EVQLVESGGGLAQAGGSLRLSCAASGRSLSNYAMGWFRQAPGKEREIVCAISRSGSITTYADSVKGRFTIAKDNAANTVYLQMNSLKPEDTAAYYCAADPMYYGIPDQNWDYWGQGTQVTVSS 5C7: 10EVQLVESGGGLVQAGDSLRLSCAASGGTFGSSHMAWFRQVPEKEREFVAAISRSGGTTYYADSVKGRCTISRDNAKNTVYLQMNSLKPEDTAVYYCAAARFWGSTSTRMDDYQYWGQGTQVTVSS 5D10: 11EVQLVESGGGLAQAGGSLRLSCAASGRSLSNYAMGWFRQAPGKEREIVCAISRSGSITTYADSVKGRFTIAKDNAANTVYLQMNSLKPEDTAAYYCAADPMYYGIPDQNWDYWGQGTQVTVSS 5E8: 12EVQLVESGGGLAQAGGSLRLSCAASGRSLSNYAMGWFRQAPGKEREIVCAISRSGSITTYADSVKGRFTIAKDNAANTVYLQMNSLKPEDTAAYYCAADPMYYGIPDQNWDYWGQGTQVTVSS 5G9: 13EVQLVESGGGLVQTGGSLRLSCAASGRTFSPYAAGWFRHAPGKEREFVTAITWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASYRWGITHEFDYWGQGTQVTVSS 6A6: 14EVQLVESGGGLAQAGGSLRLSCAASGRSLSNYAMGWFRQAPGKEREIVCAISRSGSITTYADSVKGRFTIAKDNAANTVYLQMNSLKPEDTAAYYCAADPMYYGIPDQNWDYWGQGTQVTVSS 6B2: 15EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGDSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGDVGRPDLYEYDYWGQGTQVTVSS 6B6: 16EVQLVESGGGLVQPGGSLRLSCAASGFTEDDYAMSWGRQAPGKGLEWVSAISWNGGSTYYAESMKGRFTVSRDNAKNTLYLQMNSLKSEDTAVYYCAKALDPLGVLAGTSGIYDYWGQGTQVTVSS 6E6: 17EVQLVESGGGLAQAGGSLRLSCAASGRSLSNYAMGWFRQAPGKEREIVCAISRSGSITTYADSVKGRFTIAKDNAANTVYLQMNSLKPEDTAAYYCAADPMYYGIPDQNWDYWGQGTQVTVSS 6E10: 18EVQLVESGGGLVQAGGSLRLSCAASGRTDSSYAMGWFRQAPGKEHEEVAAISWSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCGASYRWGITHEYEYWGQGTQVTVSS 6E11: 19EVQLVESGGGLAQAGGSLRLSCAASGRSLSNYAMGWFRQAPGKEREIVCAISRSGSITTYADSVKGRFTIAKDNAANTVYLQMNSLKPEDTAAYYCAADPMYYGIPDQNWDYWGQGTQVTVSS 6F6: 20EVQLVESGGGLVQPGGSLRLSCAASGSILGINAMGWYRQAPGKQRELVAVINSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADEWGDYPGQVYDYWGQGTQVTVSS 6G6: 21EVQLVESGGGLAQAGGSLRLSCAASGRSLSNYAMGWFRQAPGKEREIVCAISRSGSITTYADSVKGRFTIAKDNAANTVYLQMNSLKPEDTAAYYCAADPMYYGIPDQNWDYWGQGTQVTVSS 6H6: 22EVQLMESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKERENVAAISSSGGYTYYPDSVKGRFTISRDTAKNTVYLQMNSLKPDDTAVYYCAGTQPNFGWQLLLLQTEYDYWGQGTQVTVSS 7G6: 23EVQLVESGGGLVQAGGSLRLSCAVSGRTFSPYTMGWFRRAPGKEREFVAARTWSGGVAWYADSVKGRFTISSDNAENMVYLQMNSLKPEDTAVYYCAAKSPGRTYSPREERAYARWGQGTQVTVSS 9G6: 24EVQLVESGGGLVQAGGSLRLSCAASGRTLSDYTMGWFRQAPGKEREFVARINTGGPITSYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYNCAARLPTKMSPRDYSSYAYRGQGTQVTVSS 9H5: 25EVQLVESGGGLVQPGGSLRLSCAASGSIFNLNPMGWYRQAPGKQRELVATVTGEGRTNYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAAFWAYDDAYWGQGTQVTVSS 9E6: 26EVQLVESGGGLAQAGGSLRLSCAASGRSLSNYAMGWFRQAPGKEREIVCAISRSGSITTYADSVKGRFTIAKDNAANTVYLQMNSLKPEDTAAYYCAADPMYYGIPDQNWDYWGQGTQVTVSS 10G3: 27EVQLVESGGGLVQAGGSLRLSCADSGRTFSSYAMGWFRQAPGKEREFVAGVNWSGDSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDAAVYYCAADPVGRADLYEYDYWGQGTQVTVSS

TABLE B-4 Polypeptide sequences of the invention Name of cloneSEQ ID NO: Amino acid sequences 1E2-ALB 112EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAGISWSGSSSYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADPVGRADLYEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTESSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGAADYKDHDGDY KDHDIDYKDDDDKGAAHHHHHH6E10-ALB 113 EVQLVESGGGLVQAGGSLRLSCAASGRTDSSYAMGWERQPPGKEHEFVAAISWSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCGASYRWGITHEYEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGAADYKDHDGDYK DHDIDYKDDDDKGAAHHHHHH

TABLE B-5 Linker sequences of the invention Name of SEQ linker ID NO:Amino acid sequences 5GS 117 GGGGS 6GS 118 SGGSGGS 9GS 119 GGGGSGGGS10GS 120 GGGGSGGGGS 15GS 121 GGGGSGGGGSGGGGS 18GS 122 GGGGSGGGGSGGGGGGGS20GS 123 GGGGSGGGGSGGGGSGGGGS 25GS 124 GGGGSGGGGSGGGGSGGGGSGGGGS 30GS125 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS 126GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

1. An immunoglobulin single variable domain that can bind HGF (SEQ IDNO: 1) with a Kd of less than 50 nM.
 2. The immunoglobulin singlevariable domain of claim 1, wherein the immunoglobulin single variabledomain comprises an amino acid sequence of formula 1:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1); wherein FR1 to FR4 refer toframework regions 1 to 4 and are framework regions (FRs) of animmunoglobulin single variable domain; and wherein CDR1 is chosen fromthe group consisting of: SEQ ID NOs: 40-51, polypeptides that have atleast 80% amino acid identity with SEQ ID NOs: 40-51, and polypeptidesthat have 3, 2, or 1 amino acid difference with SEQ ID NOs: 40-51; andwherein CDR2 is chosen from the group consisting of: SEQ ID NOs: 64-75;polypeptides that have at least 80% amino acid identity with SEQ ID NOs:64-75; and polypeptides that have 3, 2, or 1 amino acid difference withSEQ ID NOs: 64-75; and wherein CDR3 is chosen from the group consistingof: SEQ ID NOs: 88-99; polypeptides that have at least 80% amino acididentity with at least one of the immunoglobulin single variable domainsof SEQ ID NOs: 88-99; and polypeptides that have 3, 2, or 1 amino aciddifference with SEQ ID NOs: 88-99.
 3. The immunoglobulin single variabledomain according to claim 2, wherein the framework regions (FRs) have asequence identity of more than 80% with the FRs of SEQ ID NOs: 7-25. 4.The immunoglobulin single variable domain according to claim 1, whereinthe immunoglobulin single variable domain comprises an amino acidsequence with the formula 1:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1); wherein FR1 to FR4 refer toframework regions 1 to 4 and are framework regions of an immunoglobulinsingle variable domain; wherein CDR1 is SEQ ID NO: 40, wherein CDR2 isSEQ ID NO: 64, 67, 69 or 72; and wherein CDR3 is SEQ ID NO: 88, 91, 93or
 96. 5. The immunoglobulin single variable domain according to claim1, wherein the immunoglobulin single variable domain comprises an aminoacid sequence with the formula 1:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1); wherein FR1 to FR4 refer toframework regions 1 to 4 and are framework regions of an immunoglobulinsingle variable domain; wherein CDR1 is SEQ ID NO: 40, wherein CDR2 isSEQ ID NO: 64; and wherein CDR3 is SEQ ID NO:
 88. 6. The immunoglobulinsingle variable domain according to claim 1, wherein the immunoglobulinsingle variable domain comprises an amino acid sequence with the formula1:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1); wherein FR1 to FR4 refer toframework regions 1 to 4 and are framework regions of an immunoglobulinsingle variable domain; wherein CDR1 is SEQ ID NO: 45, wherein CDR2 isSEQ ID NO: 69; and wherein CDR3 is SEQ ID NO:
 93. 7. A polypeptidecomprising an immunoglobulin single variable domain according toclaim
 1. 8. The polypeptide according to claim 7, wherein thepolypeptide is selected from the group consisting of polypeptides thathave an amino acid sequence with a sequence identity of more than 80%with SEQ ID NOs: 7 to
 25. 9. The polypeptide according to claim 7,wherein the polypeptide is selected from the group consisting ofpolypeptides that have an amino acid sequence with a sequence identityof more than 80% with SEQ ID NOs: 7 or
 18. 10. The polypeptide accordingto claim 7, additionally comprising an immunoglobulin single variabledomain binding human serum albumin such as e.g. Alb8 (SEQ ID NO: 115) orAlb11 (SEQ ID NO: 114).
 11. The immunoglobulin single variable domainaccording to claim 1, wherein the IC50 in an Alphascreen assay is 30 nMor lower.
 12. The immunoglobulin single variable domain according toclaim 1, wherein the IC50 in an Alphascreen assay is 3 nM or lower. 13.A nucleic acid sequence encoding an immunoglobulin single variabledomain according to claim 1 or a polypeptide-comprising theimmunoglobulin single variable domain.
 14. A pharmaceutical compositioncomprising an immunoglobulin single variable domain according to claim 1or a polypeptide comprising the immunoglobulin single variable domain;and optionally a pharmaceutically acceptable excipient.
 15. A method fortreating cancer comprising administering to a subject in need thereof aneffective amount of an immunoglobulin single variable domain accordingto claim 1 or a polypeptide comprising the immunoglobulin singlevariable domain.
 16. A method for producing an immunoglobulin singlevariable domain or a polypeptide comprising the immunoglobulin singlevariable domain, said method at least comprising the steps of: a)expressing, in a suitable host cell or host organism or in anothersuitable expression system, a nucleic acid or nucleotide sequenceaccording to claim 13; optionally followed by: b) isolating and/orpurifying said immunoglobulin single variable domain or saidpolypeptide.
 17. Method for screening immunoglobulin single variabledomains directed against HGF and in particular human HGF (SEQ ID NO: 1)that comprises at least the steps of: a) providing a set, collection orlibrary of immunoglobulin single variable domains; and b) screening saidset, collection or library of immunoglobulin single variable domains forimmunoglobulin single variable domains that can bind to and/or haveaffinity for HGF and in particular human HGF (SEQ ID NO: 1); and c)isolating the amino acid sequence(s) that can bind to and/or haveaffinity for HGF and in particular human HGF (SEQ ID NO: 1).
 18. Animmunoglobulin single variable domain that can bind HGF with a Kd ofless than 50 nM, wherein the binding of said immunoglobulin singlevariable domain to said HGF inhibits the binding of said HGF to c-Met.